System and method for automatically venting and sampling a culture specimen container

ABSTRACT

Apparatus for automated venting and/or sampling of a specimen container having a closure sealing the interior of the specimen container from the environment is disclosed. The apparatus includes a rack holding the specimen container; a venting and/or sampling device having a needle, a chamber in fluid communication with the needle and a port in fluid communication with the chamber; a robotic transfer mechanism moveable relative to the rack; a sample removal apparatus attached to the robotic transfer mechanism having gripping features for gripping the venting device. The sample removal apparatus and robotic transfer mechanism are moveable relative to the specimen container so as to automatically insert the needle of the venting device through the closure of the specimen container to thereby vent the interior of the specimen container and obtain equilibrium between the interior of the specimen container and the atmosphere and withdraw a portion of the sample from the specimen container into the venting and/or sampling device.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/800,396, filed May 14, 2010, which claims priority under 35U.S.C. §119(e) to U.S. Provisional Application No. 61/216,339, filed May15, 2009, the contents of which are incorporated by reference herein.

This application is also related to the following applications filed onthe same date as this application, the content of which is incorporatedby reference herein:

-   Docket no. 09-271-A, Ser. No. ______ “Methods for rapid    identification and/or characterization of a microbial agent in a    sample.”-   Docket no. 09-271-B, Ser. No. ______ “System for rapid    identification and/or characterizaton of a microbial agent in a    sample.”-   Docket no. 09-271-US, Ser. No. ______, “System for rapid detection    of a microbial agent in a sample and identifying and/or    characterizing the microbial agent.”

STATEMENT OF FEDERALLY SPONSORED RESEARCH

Not applicable.

BACKGROUND

This invention is directed to automated methods and apparatus forobtaining a sample from a specimen container used to culture a sampleand for venting the container to atmosphere.

Instruments currently exist on the market in the U.S. that detect thegrowth and therefore the presence of a microorganism in a blood sample.One such instrument is the BacT/ALERT 3D instrument of the presentassignee bioMérieux, Inc. The instrument receives a blood culture bottlecontaining a blood sample, e.g., from a human patient. The instrumentincubates the bottle. Periodically during incubation an opticaldetection unit in the incubator analyzes a colorimetric sensorincorporated into the bottle to detect whether microbial growth hasoccurred within the bottle. The optical detection unit, specimencontainers and sensors are described in the patent literature, see U.S.Pat. Nos. 4,945,060; 5,094,955; 5,162,229; 5,164,796; 5,217,876;5,795,773; and 5,856,175, the entire content of each of which isincorporated by reference herein. Other prior art of interest relatinggenerally to the detection of microorganisms in a biological sampleincludes the following: U.S. Pat. No. 5,770,394, U.S. Pat. No.5,518,923; U.S. Pat. No. 5,498,543, U.S. Pat. No. 5,432,061, U.S. Pat.No. 5,371,016, U.S. Pat. No. 5,397,709, U.S. Pat. No. 5,344,417, U.S.Pat. No. 5,374,264, U.S. Pat. No. 6,709,857; and U.S. Pat. No.7,211,430.

In detection instruments such as the BacT/ALERT 3D and similarinstruments, once the blood culture bottle has been tested positive formicroorganism presence, it is difficult to obtain a high level ofcharacterization of the microbial agent, or identification of thespecies of the microbial agent, due to the interference of bloodcomponents and artifacts of the disposable system (e.g., bottle)containing the sample. Therefore, current methods use a bottle or othersuitable disposable container and a related instrument for naturalgrowth and detection of a microorganism in the sample, as describedabove. Once the instrument indicates that the bottle is positive forpresence of a microbial agent, according to current methods the“positive” bottle is manually retrieved from the instrument and aportion of the sample is manually removed from the bottle and culturedon an agar plate. There are instruments in the art that automate thestreaking of a sample medium on a culture plate and incubating theplate. One such instrument is described in U.S. Pat. No. 6,617,146.After streaking, the plate is manually placed in an incubator andperiodically inspected for growth of a subculture of the microorganism.After the subculture has grown sufficiently, a sample of the culture istaken from the plate and placed in a test tube. The test tube is thenintroduced into yet another instrument for identification testing via adisposable test sample card having a multitude of individual wells. Thedisposable test cards are known in the patent literature, see e.g., U.S.Pat. Nos. 4,118,280, 3,963,355, 4,018,65; 4,116,775 and 4,038,151,5,609,828, 5,746,980, 5,766,553, 5,843,380, 5,869,005, 5,916,812,5,932,177, 5,951,952, and 6,045,758, the entire content of which isincorporated by reference herein.

The test sample card is then processed in an analytical instrument knownin the art as the VITEK 2 instrument of the assignee. The VITEK 2instrument incubates and periodically reads the wells of the test samplecard with a reader unit. Growth of the sample in one or more of thewells of the cards results in identification of the microbial agent. TheVITEK 2 instrument is described in the patent literature, see e.g., U.S.Pat. Nos. 5,762,873 and 6,086,824, the content of which is incorporatedby reference herein.

This entire process from the time of introducing the sample into theblood collection bottle to culture, detection of microorganism presence,and then identification of the microorganism by the VITEK 2 instrumenttypically takes 2-5 days. The identification steps alone, occurringafter positive bottle detection, typically occupy 1-3 of these days.

Substantial, and potentially life saving, clinical benefits for apatient are possible if the time it takes for detection andidentification of a microbial agent in a blood sample and reporting theresults to a clinician could be reduced from the current 2-5 days toless than one day. A system that meets this need has heretofore eludedthe art. However, such rapid identification and/or characterization of amicrobial agent in a biological sample such as a blood sample is madepossible by this invention.

The methods and apparatus of this disclosure are useful in their ownright; however they are particularly useful when implemented in aninstrument for rapidly identifying and/or characterizing a microbialagent as described in our prior provisional application, in co-pendingapplication Ser. No. ______, attorney docket no. 09-271-US, filed on thesame date as this application, and in embodiments disclosed herein.

SUMMARY

In one aspect, an apparatus for automated venting of a specimencontainer having a closure sealing the interior of the specimencontainer from the environment is provided. The apparatus includes arack holding the specimen container; a venting device having a needle, achamber in fluid communication with the needle and a port in fluidcommunication with the chamber; a robotic transfer mechanism moveablerelative to the rack; and a sample removal apparatus attached to therobotic transfer mechanism having gripping features for gripping theventing device. The sample removal apparatus and robotic transfermechanism are moveable relative to the specimen container so as toautomatically insert the needle of the venting device through theclosure of the specimen container to thereby vent the interior of thespecimen container and obtain equilibrium between the interior of thespecimen container and the atmosphere.

In another aspect, an automated method is disclosed for venting aspecimen container in the form of a bottle having a closure sealing theinterior of the specimen container from the environment, comprising thesteps of: holding the bottle in a rack; automatically and with the aidof robotic apparatus grasping a venting device having a needle, achamber connected to the needle and a port connected to the chamber;automatically moving the robotic apparatus so as to place the ventingdevice in a position proximate to the bottle in the rack; automaticallyinserting the needle of the venting device through the closure so as toplace the needle into the interior of the bottle, and bringing theinterior of the specimen container to equilibrium with the atmospherevia the needle, chamber and port.

In another aspect, an apparatus for automated sampling of a specimencontainer having a closure sealing the interior of the specimencontainer from the environment is disclosed. The apparatus includes arack holding the specimen container; a sampling device having a needle,a chamber in fluid communication with the needle and a port in fluidcommunication with the chamber; a robotic transfer mechanism moveablerelative to the rack; a sample removal apparatus attached to the robotictransfer mechanism having gripping features for gripping the samplingdevice; and a pneumatic system coupled to the port of the samplingdevice. The sample removal apparatus and robotic transfer mechanism aremoveable so as to automatically insert the needle of the sampling devicethrough the closure of the specimen container, the pneumatic systemoperative to draw a portion of a sample contained within the specimencontainer into the chamber of the sampling device via the needle.

In still another aspect, an automated method for sampling a specimencontainer in the form of a bottle having a closure sealing the interiorof the specimen container from the environment is disclosed. The methodincludes the step of (a) holding the bottle in a rack; (b) automaticallyand with the aid of robotic apparatus grasping a sampling device havinga needle, a chamber connected to the needle and a port connected to thechamber; (c) automatically moving the robotic apparatus so as to placethe sampling device in a position proximate to the bottle in the rack;(d) automatically inserting the needle of the sampling device throughthe closure so as to place the needle into the interior of the bottle,and (e) applying vacuum to the port of the sampling device so as to drawa portion of a sample contained in the bottle into the sampling device.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description makes reference to the appendeddrawing figures. It is intended that the embodiments and figuresdisclosed herein are to be considered illustrative and offered by way ofexample rather than restrictive. In particular, the inventive ventingand sampling apparatus of this disclosure will be described inconjunction with several embodiments of an automated instrument forrapid identification and/or characterization of a microbial agent withina specimen container. This environment for use of the apparatus of thisdisclosure is offered by way of example and not limitation.

FIG. 1 is a block diagram of an automated instrument for rapididentification and/or characterization of a microbial agent present in asample. The venting and sampling aspects of this disclosure areimplemented in the instrument of FIG. 1; however they could beimplemented in other instruments or in identification instruments withdifferent configurations.

FIG. 2 is a perspective view of one possible configuration of theinstrument shown in FIG. 1. The instrument includes a rack for holdingspecimen containers, a cassette of disposables (including samplingdevices and separation devices), a robotic transfer mechanism, sampleremoval apparatus, a separation and concentration device in the form ofa centrifuge, and a identification and/or characterization module (readstation) operating to interrogate a separation device containing aconcentrated microbial agent for identification and/or characterizationof the microbial agent. In one possible embodiment, the instrument ofFIG. 2 could be integrated with an automated detection instrument (SeeFIG. 28, 47-48 below), in which case the rack for the specimencontainers is the same structure holding the specimen containers duringthe detection operations. Alternatively, the identification and/orcharacterization instrument is located remotely from but coupled to anautomated detection instrument as shown in FIG. 47 and described in ourprior provisional application and co-pending U.S. application Ser. No.______, attorney docket no. 09-271-US, filed on the same date as thisapplication.

FIG. 3 is top plan view of the identification and/or characterizationinstrument of FIG. 2, showing the rack of positive specimen containersin one position for incubation.

FIG. 4 is a top plan view of the instrument of FIG. 2, showing the rackfor the positive specimen containers moved to a position for withdrawalof the sample from the bottle for identification and/or characterizationtesting.

FIG. 5 is another perspective view of the embodiment of FIGS. 2-4.

FIG. 6 is a perspective view of a separation device which is used inconjunction with the identification/characterization instrument ofFIG. 1. The separation device receives a portion of the sample from apositive specimen container. The microbial agent is concentrated at thebottom of a capillary tube located in the separation device in themanner described herein. The concentrated microbial agent is theninterrogated by a identification and/or characterization reading moduleto characterize and/or identify the microbial agent.

FIG. 7 is a perspective view of the separation device of FIG. 6.

FIG. 8 is a cross-sectional view of the separation device of FIGS. 6 and7.

FIG. 9 is a cross-sectional view of an end-cap which is fitted to thelower end of the separation device of FIGS. 6-8.

FIG. 10 is a cross-sectional view of the separation device of FIG. 6,showing the concentrated microbial agent in the capillary tube of theseparation device after centrifugation.

FIG. 11 is a schematic illustration of the concentrated microbial agentin the separation device of FIG. 6 being interrogated by theidentification/characterization or reading module.

FIG. 12 is a perspective view of an alternative embodiment of theseparation device of FIG. 6.

FIG. 13 is a cross-section of the separation device of FIG. 12.

FIG. 14 is an illustration of one embodiment of a disposable samplingdevice which is used within the identification and/or characterizationinstrument.

FIG. 15 is a detailed perspective view showing the operation of thesample removal apparatus in the identification and/or characterizationinstrument to pick up one of the sampling devices of FIG. 14 from acassette of disposable devices. The cassette includes a multitude of theseparation devices of FIG. 6 or 12 and a multitude of the samplingdevices of FIG. 14.

FIG. 16 is a detailed perspective view showing the operation of thesample removal apparatus to sterilize the stopper at the top of thedetection container and vent the detection container.

FIG. 17 is a more detailed illustration of the sample removing apparatusin the position shown in FIG. 16.

FIG. 18 is a detailed perspective view showing the operation of thesample removal apparatus to withdraw a portion of the sample within thedetection container into one of the disposable sampling devices of FIG.14.

FIG. 19 is a more detailed illustration of the sample removal apparatusin the position of FIG. 18.

FIGS. 20A-20C are three perspective views of the sample removalapparatus showing the operations of dispensing the sample into one ofthe separation devices and transfer the sampling device to the wastecontainer.

FIG. 21 is sequence of perspective views of the sample removal apparatusshowing the operations of transferring the separation device to theseparation and concentration station and optical interrogation of theseparation device in the identification and/or characterization module.

FIG. 22 is a more detailed illustration of the separation andconcentration station and the identification and/or characterizationmodule.

FIG. 23 is a sequence of three perspective views of the identificationand/or characterization instrument showing the operations of picking upthe separation device, transferring the separation device to the wastecontainer and placing the separation device in the waste container.

FIG. 24 is a more detailed illustration of the operation of placing theseparation device into the waste container.

FIG. 25 is a block diagram of an alternative configuration of theidentification and/or characterization instrument in which theconcentrated microbial agent is removed from the separation device andanalyzed after removal. The analysis could be performed by any one of anumber of different types of systems or units, including a moleculardiagnostic test unit, a mass spectrometry unit, or a microbialidentification test device and associated processing instrument.

FIGS. 26A-26C are a flow chart showing the steps performed in theoperation of both automatically detecting the presence of a microbialagent in a specimen container (FIG. 26A) and automatically identifyingand/or characterizing the microbial agent (FIGS. 26B and 26C).

FIG. 27 is a perspective view of a second embodiment of an instrumentfor rapid and automated identification and/or characterization of amicrobial agent in a sample.

FIG. 28 is a perspective view of the instrument of FIG. 27, showing onepossible configuration of the racks for holding the specimen containers.In the embodiment of FIG. 28, the racks include features for incubationof the specimen containers, agitation of the specimen containers, andautomated detection of microbial growth within the specimen containers.Thus, FIG. 28 shows one embodiment in which the automated detection andidentification instruments can be combined into a single instrument.

FIG. 29 is another perspective view of the embodiments of FIGS. 27 and28. A multiple axis robot is used access the specimen containers andperform the sampling operation using disposable sampling devices.

FIG. 30 is a perspective view of cassettes holding disposable samplingdevices and disposable separation devices, which can be used in theinstruments of either FIG. 2-5 or 27-29.

FIG. 31 is a perspective view of a multiple axis robot used in theembodiment of FIG. 29.

FIG. 32 is a perspective view of an alternative embodiment of adisposable sampling device, presenting a variation on the general designshown in FIG. 14.

FIG. 33 is a cross sectional view of the sampling device of FIG. 32.

FIG. 34 is a detailed perspective view of the distal end of the arm ofthe robot of FIG. 31 shown gripping the sampling device of FIG. 32.

FIG. 35 is another detailed perspective view of the distal end of thearm of the robot of FIG. 31 shown gripping the sampling device of FIG.32.

FIG. 36 is a perspective view of the pump assembly on the robot of FIG.31 which operates to provide vacuum and positive pressure to thesampling device in order to (a) withdraw a small portion of the samplefrom one of the specimen containers and (b) dispense the sample(optionally after lysing the sample) into one of the disposableseparation devices of FIGS. 6 and 30.

FIG. 37 is a perspective view of the robot of FIG. 31 performing asampling operation on one of the specimen containers using the samplingdevice of FIG. 32.

FIG. 38 is a more detailed view of the sampling operation shown in FIG.37.

FIG. 39 is a perspective view of the sampling device of FIG. 32 beingplaced into a vortexer shown in FIGS. 26 and 27 in order to facilitatelysing of cellular components in the sample withdrawn from one of thespecimen containers.

FIG. 39A is a perspective view of the vortexer having an optional coilheater around the holder of the sampling device in order to heat theholder and maintain the sample within the sampling device at 37 degreesC.

FIG. 40 is a perspective view of the sampling device of FIG. 32 beingheld by the robot hand during the vortexing operation.

FIGS. 41A and 41B are side and cross-sectional views of the vortexer andsampling device.

FIGS. 42A and 42B are cross-sectional and perspective views of a holderfor the sampling device incorporated into the vortexer.

FIGS. 43A, 43B and 43C are cross-sectional, side and perspective views,respectively, of the sampling device and the separation device prior tointroduction of the sample from the sampling device into the separationdevice. FIG. 43D is another perspective view of the sampling andseparation device, with the rubber needle sheath of the sampling deviceshown partially removed in order to show the needle of the samplingdevice.

FIG. 44 is a perspective view of the sampling device in position toinject a portion of the sample into the separation device.

FIG. 45 is a side view of the injection operation shown in FIG. 44.

FIG. 46 is a cross-section view of the sampling and separation devicesshowing the injection operation.

FIG. 46A is a detailed view of the cup and cup holder of FIG. 27,showing the cup receiving one of the separation devices; FIG. 46B showsa separation device being inserted into the cup of FIG. 46A; FIG. 46C isa cross-section of the cup holder, cup and separation device of FIG.46A.

FIG. 47 is a schematic representation of a detection instrument fordetection of a microbial agent in a biological sample coupled to anautomated identification and/or characterization instrument via aconveyor.

FIG. 48 is a schematic representation of a combined automated detectionand identification instrument which receives specimen containers in anautomated fashion e.g., via a conveyor.

FIG. 49 is a schematic representation of a combined automated detectionand identification instrument which receives specimen containersmanually from a user, e.g., via opening a door in a front panel of theinstrument. The embodiment of FIG. 49 could be implemented, for example,using the arrangement shown in FIG. 27.

FIG. 50 is schematic block diagram showing a computer system controllingthe operation of the instrument of FIGS. 27-46.

FIG. 51 is a perspective view of a second embodiment of a separationdevice which can be used in conjunction with theidentification/characterization instrument of FIG. 1. The separationdevice of this embodiment has a separate lytic chamber and a separateseparation chamber which are connected by a fluid flow-channel.

FIG. 52 is a perspective view of the separation device embodiment ofFIG. 51, showing a top plate and base plate separated from theseparation device.

FIG. 53 is a top view of the body portion of the separation deviceembodiment shown in FIG. 51.

FIG. 54 is a cross-sectional view along line A-A of the separationdevice embodiment shown in FIG. 53.

FIG. 55 is a cross-sectional view along line B-B of the separationdevice embodiment shown in FIG. 53.

FIG. 56 is a perspective view of a combined sampling and separationdevice which can be used in conjunction with theidentification/characterization instrument of FIG. 1.

FIG. 57 is a front view of the combined sampling and separation deviceshown in FIG. 56 with a pinch valve shown in the open position.

FIG. 58 is a cross-sectional view of the combined sampling andseparation device shown in FIG. 57 with the pinch valve shown in theopen position.

FIG. 59 is a front view of the combined sampling and separation deviceshown in FIG. 56 with a pinch valve shown in the closed position.

FIG. 60 is a cross-sectional view of the combined sampling andseparation device shown in FIG. 58 with the pinch valve shown in theclosed position.

FIG. 61 is a perspective view of a second embodiment of a combinedsampling and separation device.

FIG. 62 is a cross-sectional view of the combined sampling andseparation device shown in FIG. 61.

FIG. 63 is a cross-sectional view of the valve shown in FIG. 62.

FIG. 64 is a side view of a third embodiment of a combined sampling andseparation device which can be used in conjunction with theidentification and/or characterization instrument of FIG. 1.

FIG. 65 is a cross-sectional view of the combined sampling andseparation device shown in FIG. 64.

FIG. 66 is an exploded view of the combined sampling and separationdevice shown in FIG. 64.

FIG. 67 is a perspective view of a third embodiment of a combinedsampling and separation device which can be used in conjunction with theidentification/characterization instrument of FIG. 1.

FIG. 68A is a side view of the combined sampling and separation deviceshown in FIG. 67.

FIG. 68B is a cross-sectional view of the combined sampling andseparation device shown in FIG. 68A.

FIG. 69A is a side view of the combined sampling and separation deviceshown in FIG. 67.

FIG. 69B is a cross-sectional view of the combined sampling andseparation device shown in FIG. 69A.

DETAILED DESCRIPTION I. Overview

An automated instrument is described herein that provides a newarchitecture and method for automated identification and/orcharacterization of a microbial agent in a specimen sample, e.g.,biological sample. The identification and/or characterization instrument104 is shown in block diagram form in FIG. 1. Two embodiments aredescribed herein in great detail, a first embodiment described inconjunction with FIGS. 2-26 and a second embodiment described inconjunction with FIGS. 27-46. The embodiments of the instrument 104operate on a specimen container 500 (FIG. 1) containing a sample. In oneexample, the specimen container 500 is a standard culture bottle, e.g.,a blood culture bottle, for containing a specimen sample therein, e.g.,a blood sample.

The venting and sampling aspects of this invention are included asfeatures of the instrument 104 as will be apparent from the followingdiscussion.

In general, any type of sample that may contain a microbial agent, e.g.,bacterium, fungi or yeast species, can be tested in the instrument 104such as for example biological samples. For example, the specimen samplecan be a clinical or non-clinical sample suspected of containing one ormore microbial agents. Clinical samples, such as a bodily fluid,include, but not limited to, blood, serum, plasma, blood fractions,joint fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastriccontents, vaginal secretions, tissue homogenates, bone marrow aspirates,bone homogenates, sputum, aspirates, swabs and swab rinsates, other bodyfluids, and the like. Non-clinical samples that may be tested include,but not limited to, foodstuffs, beverages, pharmaceuticals, cosmetics,water (e.g., drinking water, non-potable water, and waste water),seawater ballasts, air, soil, sewage, plant material (e.g., seeds,leaves, stems, roots, flowers, fruit), blood products (e.g., platelets,serum, plasma, white blood cell fractions, etc.), donor organ or tissuesamples, biowarfare samples, and the like.

One possible configuration for the instrument 104 of this disclosure isin a combined system which integrates detection of a microbial agent ina specimen container with automated identification and/orcharacterization of the microbial agent. Such a combined approach isdescribed in the prior provisional application and in co-pendingapplication Ser. No. ______, attorney docket no. 09-271-US, filed on thesame date as this application. This combined approach is also describedin conjunction with the embodiment of FIG. 27.

In this configuration, a specimen container 500 (FIG. 1) is inoculatedwith a specimen sample (e.g., clinical or non-clinical sample) andloaded/unloaded into/out of an automated detection instrument 102 (e.g.FIG. 47). After a sufficient time interval to allow naturalamplification of microorganism (this time interval varies from speciesto species), the specimen container is tested within the detectioninstrument 102 for the presence of a microorganism. The testing occurson a periodic basis so that as soon as a specimen container is testedpositive it can be transferred to the identification and/orcharacterization instrument 104 for further analysis of the specimensample.

Detection can be accomplished using a variety of technologies such asthe colorimetric sensor described in the patent literature (see U.S.Pat. Nos. 4,945,060; 5,094,955; 5,162,229; 5,164,796; 5,217,876;5,795,773; and 5,856,175). Detection could also be accomplished usingintrinsic fluorescence of the microorganism, detection of changes in theoptical scattering of the media, or detection in the generation ofvolatile organics in the media or headspace. These techniques are knownin the art and described in previously cited patent literature in theBackground section of this document.

Once a specimen container 500 is detected as positive in the automateddetection instrument 102 (see FIG. 47), the detection instrument 102will notify the operator through an indicator (e.g., visual prompt), orvia a notification at the user interface display, or by other means. Thesystem may be set up to automatically analyze a positive specimencontainer or require end user acknowledgement prior to sample analysisin the identification/characterization instrument 104 described below.With automatic characterization, it would be possible to notify thephysician immediately via electronic means of the results from theidentification/characterization system.

Once a specimen container is determined to be positive in the detectioninstrument 102, the positive specimen container is handed off ortransferred to the identification and/or characterization instrument 104described below. See FIG. 47. The manner in which this is accomplishedcan vary widely depending on the physical configuration of the detectionand identification/characterization instruments 102 and 104. One exampleof how this can be accomplished is described below in conjunction withFIG. 27.

Referring now in particular to FIG. 1, a specimen container or bottle500 is received in the identification and/or characterization instrument104, either in an automated or manual fashion. The manner in which thisoccurs is not particularly important and can vary widely depending onthe configuration of the instrument 104. The specimen container 500 isplaced in a suitable holding structure or rack 1906 in theidentification and/or characterization instrument 104. FIGS. 2, 5, 27and 28 show several possible configuration for the holding structure1906. The holding structure 1906 is typically adapted for holding amultitude of specimen containers 500. The holding structure 1906 has afacility for rotating the specimen containers to inclined positionsabove and below horizontal to facilitate venting and sample removal, asdescribed below and optionally agitation of the sample and therebypromoting microbial growth. In an alternative configuration, thepositively declared specimen container could remain in the racks withinthe detection instrument 102 and the sampling could occur directly fromthe detection instrument.

The identification and/or characterization instrument 104 includes asample removal apparatus 1912 which holds or grasps a disposable ventingand/or sampling device 1902. Together, they operate to vent the specimencontainer and remove a test sample (i.e., a portion of the specimensample in the positive specimen container 500) and subsequently add theportion to a separation device 1904 (see FIGS. 6-11). The separationdevice 1904 can take several forms, and one configuration is describedherein in which the separation device includes a reservoir (FIG. 8, item2602) for receiving the sample and a capillary tube 2604 connected tothe reservoir 2602. The identification/characterization instrument 104further includes a separation and/or concentration station 1916,optionally in the form of a centrifuge, which operates on the separationdevice 1904 so as to separate the microbial agent from other componentsin the test sample and concentrate the microbial agent within theseparation device 1904. In one example, the microbial agent isconcentrated in the form of a pellet or pellet-like mass in the bottomof the capillary tube 2604 of the separation device 1904. Theidentification/characterization instrument further includes aidentification and/or characterization module or read station (FIG. 1,1918) which interrogates the concentrated microbial agent to identifyand/or characterize the microbial agent.

The instrument 104 receives a cassette 1900 of disposables. Thedisposables are of two types: (1) sampling devices 1902 for venting andremoving a test sample from the specimen container 500, and (2)separation devices 1904 which receive a portion of the sample from thecontainer 500 via the venting and/or sampling device 1902 and in whichthe microbial agent in the test sample is concentrated. In alternativeconfiguration of the instrument the functions of the venting/and orsampling device 1902 and the separation device 1904 are combined into asingle disposable device as shown in FIGS. 51-60 in which case thecassette 1900 will only include a multitude of the combined sampling andseparation devices.

The instrument 104 further includes a robotic transfer mechanism 1910which operates to access the disposables 1902 and 1904, positivespecimen containers 500 held in the holder or rack 1906, a wastecontainer 1908, the separation and concentration device 1916, and theidentification module 1918. The robotic transfer mechanism 1910 may alsooperate to receive a positive specimen container from a separatedetection instrument, and load the positive specimen container into theholding structure or rack 1906. The robotic transfer mechanism 1910accesses the waste container, separation and concentration station 1916,identification module 1918 and other modules or components in theinstrument 104 as necessary to perform the functions described below.The manner of construction of the transfer mechanism 1910 can varywidely depending on the configuration of the instrument 104.

The sample removal apparatus 1912 is preferably incorporated into, orcoupled to, the robotic transfer mechanism 1910 as indicated by thedashed lines 1913. The apparatus 1912 further includes robot grippingand handling mechanisms to grasp one of the venting and sampling devices1902, the separation device 1904 and/or the specimen container 500. Thesample removal apparatus 1912 is connected to a pneumatic system 1914which enables robotic gripping functions. The pneumatic system 1914 mayinclude a vacuum pump, as described in the second embodiment below. Thevacuum pump operates to provide vacuum to the venting and samplingdevice 1902 to draw a sample from the specimen container 500 and providepositive pressure to the sampling device 1902 to inject the sample fromthe sampling device 1902 into the separation device 1904. These aspectsof the identification instrument 104 will all be described in greaterdetail below.

In one embodiment, the identification module 1918 includes a lightsource (e.g., an excitation light source) which illuminates theconcentrated microbial agent in the separation device 1904. In responseto the illumination, the concentrated microbial agent emits a detectablefluorescence signal, i.e., intrinsic fluorescence, as described below.In addition, the illumination of the concentrated microbial agent by thelight source will generate a reflectance signal or Rayleigh scatteringsignal; this signal is of the same wavelength of the excitation lightand provides additional information about the absorption of themicrobial agent. The reflectance signal may also provide the basis ofnormalization of the fluorescence data The configuration of theidentification module 1918 includes a means for spatially dispersing thereflectance/fluorescence spectrum, which may take the form of aspectrometer. These fluorescence and reflectance signals (spectrum) arecaptured by a sensor array 1920 which generates signals supplied to acomputer 1924. The computer executes algorithms to process thereflectance/fluorescence signals and responsively identifies and/orcharacterizes the microbial agent. In one embodiment, a reportcontaining the identification or characterization result is sent to anoutput device 1926 (e.g., display or associated computer workstation,pager, cell phone or e-mail server). The results can include clinicalgram type, direct identification of the microbial agent (e.g., to thegenus or species level in a taxonomic hierarchy), or other clinicalinformation regarding the microbial agent in the sample.

First Embodiment FIGS. 1-26

-   -   Sample removal apparatus and sampling from the specimen        container (e.g., blood culture bottle 500) (FIGS. 1-5, 15-16,        items 1910 and 1912)

A sample removal apparatus, in the form of a sample head 1912, retrievesa sampling device 1902 (disposable) from a cassette 1900 of such devices(FIGS. 1, 5). This sampling device 1902 (FIG. 14, see also FIGS. 32, 33)may take the form of a sterile sheathed needle or other means to piercea stopper or other closure member in the specimen container 500 and ventthe specimen container (if necessary) so as to equilibrate the bottlepressure with atmospheric pressure. The sampling device (FIG. 14, 32,1902) includes a sampling container or chamber 3204 to hold thewithdrawn test sample. The test sample will include a portion of thespecimen sample and any culture media present. Another possibleembodiment is that the sampling device contains the sterile sheathedneedle and is directly connected to or incorporated into to theseparation device (i.e., a combined sampling and separation device, seeFIGS. 60-78). The sample removal apparatus 1912 may optionally includefeatures to decontaminate the surface of the bottle prior to sampling(if necessary).

The robotic transfer mechanism 1910 (FIG. 5) can be moved in threemutually orthogonal translation axes in addition to one rotational axisaround one of the orthogonal translation axes so as to be able toposition the sample removal apparatus 1912 opposite the access point(e.g. stopper, or septum) of each specimen container/bottle 500 whilethe bottle is held in the racks 2310 of the specimen container holder1906. Alignment of a sheathed needle 3202 of the sampling device 1902 tothe bottle access point can either be accomplished by a docking featurebuilt-in to the container 500, a vision system (e.g., camera) or usingpre-programmed dimensional coordinates and precision motion controllingof the robot transfer mechanism 1910. The bottle 500 is preferably firsttilted upward so that the space below the access point or stoppercontains the headspace gases and not liquid media. The rationale forthis step is that the container should first be vented so that thepressure in the bottle is close to atmospheric pressure. This wouldprevent venting of aerosols from the bottle and excess fluid transferand overfill and possible spillage in the case of a bottle over-pressuresituation.

Similarly, if the culture has not produced significant by-products (e.g.headspace gases) or the microorganism is not a “gas producer”, therewill be an under-pressure condition or the pressure inside the bottlewill be below atmospheric pressure which would make sampling difficult.The aseptic venting will equilibrate the pressure so that a fluid samplecan be removed from the bottle.

After proper venting, the bottle 500 is tilted so that the access portof the bottle is oriented downwards and a liquid sample can betransferred to the sampling device 1902. The sampling device withdrawsfor example a 0.5 ml, 1.0, or 2.0 ml sample of blood/media from thespecimen container. Alternatively, a positive displacement syringe likedevice could be developed to provide sampling of specimen containersover a wide range of vacuum or pressure conditions.

The functions of venting and sampling could be performed by separatedisposable devices 1902, each having the configuration of FIGS. 14 and32. Alternatively, the functions of venting and sampling could beperformed by a device that includes the separation and concentrationfunction, see FIGS. 51-60 and the discussion below.

Optional Lysis of Components in the Test Sample

After the test sample has been withdrawn from the specimen container500, any cellular components contained therein (e.g., blood cells) mayneed to be lysed so that they do not interfere with separation andidentification/characterization processes described below. The optionallysis step can be performed using a lysis buffer (which is a pH balancedsurfactant solution) or can be accomplished using sonication. Bothapproaches cause disruption of the blood cell walls. The lysis operationcan be performed by adding the lysis buffer to the disposable samplingdevice 1902 either off-line or within the identification and/orcharacterization instrument 104. Alternatively, the lysis buffer can bemixed with the blood/media sample during the loading of the sample intothe separation device 1904. After the lysis buffer and blood/mediasample are combined, some amount of agitation or mixing needs to beperformed to ensure the lysis buffer contacts the blood cells and cellwall rupture occurs. In one possible embodiment, the robotic transfermechanism may move up and down or otherwise to accomplish this mixing.In another embodiment, a mixing station (e.g., a vortexer as describedin the second embodiment below) can be included in the instrument 104for accomplishing this mixing.

As an alternative, the separation device 1904 could have twocompartments separated by a thermoresponsive gel or other separationmaterial that would allow the lysis buffer and the blood/media mixtureto be combined, then pass through into the microorganism separationdevice.

Another approach could incorporate a filter to collect themicroorganisms on a surface and then resuspend the microorganisms intoan inoculum for testing.

It is envisioned the multiple separation devices 1904 could be providedin a format such as a cartridge, cassette, disk or strip to facilitateease of user loading the system.

-   -   Separation and/or concentration station (FIGS. 1, 21, item 1916)        and separation device (FIG. 1, 6-11, 21, item 1904)

After withdrawal of the specimen from the specimen container, and afteroptional lysing of the cellular components (e.g., blood cells) in thesampling device 1902, the sample is then injected or otherwiseintroduced into one of the separation devices 1904. A microbial agentpresent in the sample is separated from other components andconcentrated into a pellet or pellet-like mass within the separationdevice 1904.

The details of the separation and/or concentration of the microorganismin the separation device (1904) are described in related patentapplications incorporated by reference into this applicationhereinabove, but the basic method will be described below. Theseparation is accomplished using a density solution or density cushionfiltration. In one embodiment, the separation device 1904 is preloadedwith the density cushion. Separation and concentration occurs by meansof centrifugation of the separation device 1904.

The separation device 1904 (FIGS. 6-11) itself can take the form of acapillary tube design with a 1-2 mm diameter internal cross sectioncapillary tube filled with the density solution. Above this capillarytube region is a fluted structure that opens up (i.e., opens to a largercross sectional area) to provide a reservoir for the blood/media sampleand the density solution. The bottom surface of the separation device ismade of a material that has very good ultraviolet and visible lighttransmission. The top of the structure has a lid that is applied beforecentrifugation. In alternative configurations the separation devicecould be illuminated from the side in which case the lower portion ofthe separation device is made from a material that has very goodultraviolet and visible light transmission; the cross-sectional shape ofthe capillary tube may be circular or square.

The mixed or lysed sample contents (lysis buffer and test sample) areloaded into the separation device 1904 (see FIGS. 20A-C and thedescription below) by means of injecting the sample from the samplingdevice 1902 into the separation device 1904. The density solution iseither loaded into the separation device 1904 on-line in theidentification and/or characterization instrument 104 or, morepreferably the separation device 1904 is shipped pre-filled with thedensity solution. After the mixed or lysed sample is loaded into theseparation device 1904 and the device 1904 capped, the separation device1904 is loaded into a centrifuge 1916. Alternatively, this lid isconfigured with a septum. The sample can be added to the device 1904 bypiercing the septum, preventing the need for lid removal andreplacement. The centrifuge is activated and spun, e.g. for severalminutes at high rpm. This action causes the microbial agent (which isnot lysed) to pass through the density solution and concentrate at thebase of the capillary tube in the separation device 1904 into a pelletor pellet-like mass in the very bottom of the tube (see FIG. 10,concentrated microbial agent pellet 2804). In one embodiment, the device1904 loaded with density cushion is centrifuged prior to loading of thetest sample to remove any air bubbles or the like that may otherwiseinterfere with the separation and/or concentration step.

Again, a combined sampling and separation device could be used for bothremoving a test sample from the specimen container and concentration ofthe microbial agent; see FIGS. 51-60 and the discussion below.

-   -   Identification/characterization module (read station) for        microbiological identification and/or characterization (FIG. 1,        21, 22 item 1918)

After the separation device 1904 has been centrifuged as describedabove, the centrifuge 1916 can be rotated so that the separation device1904 is in a reading position wherein a identification and/orcharacterization module (read station) 1918 can interrogate theseparated and/or concentrated microbial agent (FIG. 10, pellet 2804).Alternatively, the separation device 1904 can be removed from thecentrifuge by the robotic transfer mechanism 1910 and placed in a readstation in a separate location.

In one form, the read station 1918 includes an optical reader assemblyfor interrogating the concentrated microbial agent (pellet) within theseparation device 1904. Since the microorganism/microbial agent in theblood/media sample is forced to the bottom surface of the capillary tubein the separation device 1904 (see FIGS. 10 and 11), the microbial agentwill be in contact with the bottom surface. In one possibleimplementation, the optical reader assembly observes the fluorescencesignal (e.g., intrinsic fluorescence signal) emitted from theconcentrated microbial agent due to illumination from an excitationlights source.

The fluorescence signal (e.g., intrinsic fluorescence) results fromexcitation by a UV, visible spectrum or IR light source (see FIG. 11).The light sources could be continuum lamps such as a deuterium or xenonlamp for UV and/or a tungsten halogen lamp for visible/IR excitation.Since these light sources have a broad range of emission, the excitationband can be reduced using optical bandpass filters. Other methods foremission wavelength spectral width that may be utilized include anacousto-optic tunable filter, liquid crystal tunable filter, an array ofoptical interference filters, prism spectrograph, and still others.Alternatively, lasers are available in discrete wavelengths from theultraviolet to the near infra-red; additionally many multiplexingmethods are known to those skilled in the art,

Alternatively, light emitting diodes can be used as narrowbandexcitation light sources. LED's are available from a peak wavelength of240 nm to in excess of 700 nm with a spectral width of 20-40 nm. Thesame methods for the reduction of spectral width can be incorporatedwith the LED's to improve discrimination between excitation and emissionspectra.

The emission from the sample may be measured by any suitable means ofspectral discrimination, most preferably employing a spectrometer. Thespectrometer may be a scanning monochromator that detects specificemission wavelengths whereby the output from the monochromator isdetected by a photomultiplier tube and/or the spectrometer may beconfigured as an imaging spectrograph whereby the output is detected byan imaging detector array such as a charge-coupled device (CCD) detectorarray. In one embodiment, a discriminator allows the observation of thefluorescence and/or scattering signal by a photodetection means (such asa photomultiplier tube, avalanche photodiode, CCD detector array, acomplementary metal oxide semiconductor (CMOS) area sensor array and/orelectron multiplying charge coupled device (EMCCD) detector array (FIG.1, item 1920). An optical lens system (2904 in FIG. 11) in front of thesensor array will magnify the 0.78-2.0 mm² area forming the bottom ofthe capillary tube 2604 so that it fills the frame of the sensor array.Alternatively, coupling between the disposable separation device 1902and the optical fiber is direct optical fiber coupling with no lenssystem; the optical fiber probe is a six around one configuration at thedistal end, with the proximal end having a linear configuration foremission fibers to couple into the entry slit of a spectrometer.Fluorescence signal strength at several different wavelengths areacquired and saved in a computer memory.

An alternative configuration is to reduce the capillary tube 2604 toless than 1 mm in diameter to account for low biomass samples.Furthermore, the geometry of the capillary area may take other shapes,such as a rectangular-shaped internal cross-section. Another optionalembodiment is to configure the reading of the capillary tube from theside instead of from the bottom. There are two possible benefits todoing so: (1) avoid debris or fibers that sediment to the base of thecapillary tube and (2) provide the opportunity to optically identify thepresence of polymicrobic agents. A rectangular shaped capillary tube maybe preferred for this side read application.

The identification and/or characterization module 1918 includes acomputer (FIG. 1, item 1924) that operates on the fluorescence signalstrength measurements which are stored in memory. The measurements arecompared to experimentally-determined fluorescence spectra measurementsfor different types of microorganisms (i.e. Gram positive, Gramnegative, yeast, etc.) that are also stored in memory. The computerexecutes a classification algorithm and generates a classificationresult for the microbial agent, e.g., gram classification, gram family,and species. In one configuration, further analysis of the spectra ofthe captured intrinsic fluorescence signal is accomplished so thatspecies identification and/or characterization or at least the top threeprobabilities for species identification is achieved. Details on themethods executed by the computer are not particularly important to theinstant invention and therefore a detailed discussion is omitted for thesake of brevity.

-   -   Disposal of sampling device 1902 and separation device 1904        (FIGS. 1, 23, item 1908)

After the test sample is injected from the sampling device 1902 into theseparation device 1904, the sampling device 1902 is discarded into abiowaste container 1908 within the identification and/orcharacterization instrument 104. After the reading of the separationdevice 1904, the separation device 1904 is also discarded in thebiowaste container 1908. The biowaste container is periodically removedfrom the identification/characterization instrument and emptied, andthen replaced into the identification/characterization instrument.

User Interface

The identification instrument 104 preferably includes a user interface(not shown) which provides an operator with status information regardingspecimen containers loaded into the identification instrument. The userinterface may include some or all of the following features:

-   -   Touch screen display    -   Keyboard on touch screen.    -   System status    -   Positives alert    -   Communications to other systems (DMS, LIS, BCES & other        detection or identification Instruments).    -   Specimen Container status    -   Retrieve specimen containers    -   Visual and audible Positive Indicator    -   USB access (back ups and external system access).    -   Remote Notification of Identification and/or Characterization        Results, System Status and Error Messages

The particular appearance or layout of the user interface is notparticularly important.

The identification results are sent to an output device 1926 (FIG. 1),which may be a computer memory, instrument display, printer, pager, cellphone, personal digital assistant, e-mail server, or other device. Theresults will typically include one or more of the following: clinicalgram type of the microbial agent, identification and/or characterizationof the species of the microbial agent, or other clinical information.

Specimen Container 500

The specimen container 500 shown in FIG. 1 is designed to hold andcontain a sample and may take the form of a standard culture bottle,e.g., blood culture bottle. Preferred embodiments of the bottleincorporate a bar code (FIG. 1) for automated reading of the bottle 500within the identification/characterization instrument 104 or off-lineequipment. The bottle 500 includes a stopper (not shown) sealing thecontainer from the environment having a pierceable septum. Optionally,where the bottle is used for both detection and automatedidentification, the bottle includes a colorimetric sensor formed orplaced in the bottom of the bottle for purposes of colorimetricdetection of the presence of microbial growth in the bottle 500.Specimen containers of the type shown in FIG. 1 are well known in theart and described in the patent literature cited in the Backgroundsection of this document, therefore a further description isunnecessary.

The configuration of the bottle is not particular important and theinventive system and methods can be adapted to a variety of containersfor containing a sample. Thus, the present description of blood culturespecimen containers is offered by way of example and not limitation.

II. Detailed Description of First Embodiment FIGS. 1-26

FIG. 2 shows one possible configuration of theidentification/characterization instrument 104, including the cassetteof disposables 1900, a rack or holder 1906 for positive specimencontainers, a waste container 1908, a robotic transfer mechanism 1910, asample removal apparatus 1912 which is attached to or coupled to therobotic transfer mechanism 1910, a separation and concentration station1916, and the identification and/or characterization module 1918. FIG. 3is a top plan view of the arrangement of FIG. 2. The holder 1906includes three racks that are oriented in one position for incubationand receiving new positive specimen containers, e.g., from a remotedetection instrument or a manual loading door. In FIG. 4, the racks aremoved to a position for sample removal from the specimen containers, andloading of the sample into the separation device 1904.

FIG. 5 is a perspective view of the identification/characterizationinstrument in the position of FIG. 4, showing theidentification/characterization instrument 104 in further detail. Theholder 1906 includes three separate racks 2310, each holding twentyspecimen containers 500. The racks 2310 are rotatable as a unit aboutthe horizontal axis to tilt the specimen containers into upwardorientation (shown in FIG. 5) for purposes of venting the specimencontainers and to a downward orientation (see FIG. 15) for sampleremoval.

The robotic transfer mechanism 1910 includes vertical guide rails 2320and a horizontal guide rail 2324. The sample removal apparatus 1912 ismoved from left to right and up and down by means of collars connectedto the guide rails and a motor and belt driving subassembly (not shown,but conventional). Thus, the sample removal apparatus 1912 can move toany of the bottle positions in the three racks 2310, when the specimencontainers are in either the upward or downward orientation. The sampleremoval apparatus 1912 can further move fore and aft by sliding alongthe guides 2322.

FIG. 5 also indicates that the instrument 104 includes electronics 2300,which includes a computer 1924 for processing fluorescence measurements,a memory 2302 storing results of the analysis and a further memory orprocessing units 2304 for storing program code for operation of theidentification/characterization instrument 104. The electronics 2300 arepreferably located behind suitable panels, which are not shown.

Cassette of Disposables

FIG. 5 shows a cassette 1900 of disposable devices which is loaded intothe identification/characterization instrument 104. The cassette 1900includes a multitude of sampling devices 1902 and separation devices1904.

The separation device 1904 is shown in FIGS. 6-11. Referring to theseFigures, the separation device consists of a body 2402 that defines areservoir 2602 and a capillary tube 2604 which is connected to thereservoir 2602. The body 2402 defines an axis 2608 and the capillarytube 2604 is oriented along the axis 2608. A first end of the capillarytube 2610 is connected to the reservoir 2602 and the second end 2612 ofthe capillary tube communicates with a tube portion 2702 of an end piece2502. The reservoir is accessed via a removable cap 2404 that threadsonto threads 2502 formed at the top portion of the body 2402. The lowerportion of the body 2402 is closed off by an end piece 2502 which isaffixed to the body by means of a ridge 2704 fitting into acorresponding recess 2606 in the body and welding or use of an adhesive.The bottom wall 2506 of the end piece 2502 is of reduced thickness asindicated in FIG. 8. The end piece incorporates a capillary tube 2702which is aligned with the capillary tube 2604 of the body 2402. The body2402 proximate to the second end of the capillary tube is made from anoptically transparent material; in the embodiment of FIG. 7 the endpiece 2502 is optically transparent for facilitating opticalinterrogation of the concentrated microbial agent 2804 located at thebottom of the capillary tube 2604. The separation device 1904 is loadedwith a density solution or “cushion” 2802 (FIG. 10), either preloadedwith the material or less preferably the material is added to theseparation device within the identification/characterization instrument.

FIGS. 12 and 13 show an embodiment of the separation device 1904 inwhich the body of the separation device 1904 is a one-piececonstruction. Walls 3002 provide support for the lower portion of thecapillary tube 2602. The body proximate to the lower portion of thecapillary tube 2604 is made from an optically transparent material.

FIG. 11 shows the operation of interrogation of concentrated microbialagent 2804 within the separation device 1904, an operation performed bythe identification module 1918 of FIGS. 1 and 5. Light from a lightsource passes along an optical fiber 2902 and is directed by lens system2904 to the base of the separation device 1904. The light stimulates theproduction of fluorescence from the microbial agent 2804 and thefluorescence is directed via the optical fiber 2906 through the lens2904 and fiber 2902 to a CCD sensor array (1920, FIG. 1).

The sampling device 1902 is shown schematically and parts not to scalein FIG. 14. The device 1902 can take the form of a syringe-like devicehaving a body 3200 defining a chamber 3204 and a sheathed needle 3202.The chamber 3204 may be pre-loaded with a selective lysis buffer 3206.The top of the chamber 3204 is sealed. The chamber may have a port 3208which allows the sampling device to be connected to a vacuum orpneumatic unit to facilitate venting or sampling of a sample from thebottle 500. The lysis buffer 3206 can be pre-loaded into the samplingdevice 1902, or it may be loaded into the device 1902 in the instrumentat the time of use.

The lysis buffer loaded into the sampling device 1902 may be tailored tothe specie(s) expected to be found. In one possible configuration,several reservoirs of selective lysis buffers are present in theinstrument 104 and one of the lysis buffers is loaded into the samplingdevice 1902 at the time of use.

Sample Removal Apparatus (Sampling Head) 1912

The sample removal apparatus 1912 of FIGS. 1 and 5 operates to remove aportion of the biological sample in the positive container 500 from thecontainer 500 and add the portion to a separation device 1904 obtainedfrom the supply of separation devices 1900. The physical configurationof the sample removal apparatus 1912 can take a variety of forms,depending on the configuration of the specimen containers, the samplingdevice, and the separation device. In the illustrated embodiment thesample removal apparatus 1912 takes the form of articulating fingersthat open and close so as to grasp the sampling device 1902 and theseparation device 1904. The sample removal apparatus 1912 is moved tothe required position for sampling and loading into the separationdevice by means of operation of the robotic transfer mechanism 1910.

Venting and Sampling

With reference to FIG. 15, the sample removal apparatus 1912 is moved toa position where it is placed directly over one of the sampling devices1902 in the cassette 1900. The fingers of the sample removal apparatus1912 grip the sampling device 1902 and the apparatus 1912 is raisedupwards, removing the sampling device 1902 from the cassette 1900. Asshown in FIG. 16, the specimen containers 500 are tilted upwards. Thestopper at the top of the bottle is sterilized using UV light or adisinfecting agent (e.g., bleach or alcohol). As shown in FIG. 17, thebottle is vented by introducing the needle 3202 (FIG. 14) of thesampling device through a pierceable septum in the stopper of the bottle500, equalizing the pressure within the interior of the bottle to thatof ambient conditions. The port 3208 of the sampling device may beconnected to the pneumatic system (1914, FIG. 1) during this process,e.g., a rolling diagram pump 1710 as shown in the second embodimentbelow.

As shown in FIGS. 18 and 19, the racks 2310 are then rotated to thedownward orientation. The sample removal apparatus 1912, in conjunctionthe pneumatic system, withdraws a test sample from the bottle 500 intothe sampling device 1902.

Lysis

The sampling device 1902 is optionally loaded with approximately 1 ml ofa lysis buffer 3206 (FIG. 14). In this embodiment, an approximately 2 mltest sample removed from the bottle 500 and mixed with the lysis bufferin the sampling device 1902, e.g., by agitation of the device 1902 afterloading into the sampling device 1902. The lysis operation is selectiveto non-microorganism component (e.g., the blood cells), i.e., themicrobial agent cells are not lysed.

The lysis buffer 3206 selectively lyses undesired cells (i.e.,non-microorganism cells) that may be present in the sample, e.g., bloodcells and/or tissue cells. The selective lysis of non-microorganismcells permits separation of microorganisms from other components thatmay be present in the sample. Accordingly, the lysis solution is onethat is capable of selectively lysing cells, e.g., non-microorganismcells (e.g., by solubilizing eukaryotic cell membranes). The lysissolution may comprise one or more detergents, one or more enzymes, or acombination of one or more detergents and one or more enzymes.

Useful detergent may include one or more non-denaturing lytic detergent,such as Triton® X-100 Triton® X-100-R, Triton® X-114, NP-40, Genapol®C-100, Genapol® X-100, Igepal® CA 630, Arlasolve™ 200, Brij® 96/97,CHAPS, octyl β-D-glucopyranoside, saponin, and nonaethylene glycolmonododecyl ether (C12E9, polidocenol). Optionally, denaturing lyticdetergents can be included, such as sodium dodecyl sulfate,N-laurylsarcosine, sodium deoxycholate, bile salts,hexadecyltrimethylammonium bromide, SB3-10, SB3-12,amidosulfobetaine-14, and C7BzO. Optionally, solubilizers can also beincluded, such as Brij® 98, Brij® 58, Brij® 35, Tween® 80, Tween® 20,Pluronic® L64, Pluronic® P84, non-detergent sulfobetaines (NDSB 201),amphipols (PMAL-C8), and methyl-β-cyclodextrin. In one embodiment,polyoxyethylene detergent detergents may be preferred. Thepolyoxyethylene detergent can comprise the structure C₁₂₋₁₈/E₉₋₁₀,wherein C12-18 denotes a carbon chain length of from 12 to 18 carbonatoms and E9-10 denotes from 9 to 10 oxyethylene hydrophilic headgroups. For example, the polyoxyethylene detergent can be selected fromthe group consisting of Brij® 97, Brij® 96V, Genapol® C-100, Genapol®X-100, nonaethylene glycol monododecyl ether (polidocanol), or acombination thereof. ethylenediaminetetraacetic acid (EDTA)

The lysis solution may also comprise one or more enzymes. Enzymes thatcan be used in the lysis solutions include, without limitation, enzymesthat digest nucleic acids and other membrane-fouling materials (e.g.,proteinase XXIII, DNase, neuraminidase, polysaccharidase, Glucanex®, andPectinex®).

In another embodiment, one or more additional agents can be used,including for example, reducing agents such as 2-mercaptoethanol (2-Me)or dithiothreitol (DTT), stabilizing agents such as magnesium, pyruvate,and humectants, and/or chelating agents such asethylenediaminetetraacetic acid (EDTA). The lysis solution can bebuffered at any pH that is suitable to lyse the desired cells, and willdepend on multiple factors, including without limitation, the type ofsample, the cells to be lysed, and the detergent used. In someembodiments, the pH can be in a range from about 2 to about 13, e.g.,about 6 to about 13, e.g., about 8 to about 13, e.g., about 10 to about13. Suitable pH buffers include any buffer capable of maintaining a pHin the desired range, e.g., about 0.05 M to about 1.0 M CAPS.

Dispense into Separation Device 1904 and Separation/Concentration

As shown in FIGS. 20A and 20B, the sample removal apparatus 1912 carriesthe sampling device 1902 (loaded with a mixed lysis buffer and samplesolution) to the position of one of the separation devices 1902 in thecassette 1900. The sample removal apparatus pierces the cap of theseparation device 1904 with the needle 3202 of the sampling device 1902and injects 0.5 to 1.0 ml of the sample+lysis buffer mixture into thereservoir of the separation device 1904. The dispensing could also beperformed after uncapping the separation device 1904 and recapping theseparation device 1904 after recapping. The sample removal apparatusthen transfers the sampling device 1902 to the waste container 1908 asshown in FIG. 20C and deposits it into the waste container.

In one embodiment, the separation is carried out by a centrifugationstep in which the sample (e.g., a lysed sample) is placed on top of anapproximately 1 ml liquid phase density cushion 2802 (FIG. 10)previously loaded in the separation device 1904 and the device 1904 iscentrifuged under conditions (e.g., 10,000 g) which allow themicroorganisms to be isolated and concentrated (e.g., the microorganismsform a pellet or pellet-like mass at the bottom and/or sides of theseparation device 1904). “Density cushion” refers to a solution having ahomogenous density throughout. The density of the cushion is selectedsuch that the microorganisms in the sample pass through the cushionwhile other components of the sample (e.g., blood culture broth, celldebris) remain on top of the cushion or do not pass all of the waythrough the density cushion.

The material for the density cushion 2802 can be any material that hasthe appropriate density range for the methods of this invention. Ingeneral, the density of the cushion is in the range of about 1.025 toabout 1.120 g/ml. In one embodiment, the material is colloidal silica.The colloidal silica may be uncoated (e.g., Ludox® (W.R. Grace, CT)) orcoated, e.g., with silane (e.g., PureSperm® (Nidacon Int'l, Sweden) orIsolate® (Irvine Scientific, Santa Ana, Calif.)) or polyvinylpyrrolidone(e.g., Percoll™, Percoll™ Plus (Sigma-Aldrich, St. Louis, Mo.)). Thecolloidal silica may be diluted in any suitable medium to form theproper density, e.g., balanced salt solutions, physiological saline,and/or 0.25 M sucrose. Suitable densities can be obtained with colloidalsilica at a concentration of about 15% to about 80% v/v, e.g., about 20%to about 65% v/v. Another suitable material for density cushions is aniodinated contrast agent, e.g., iohexol (Omnipaque™ NycoPrep™, orNycodenz®) and iodixanol (Visipaque™ or OptiPrep™). Suitable densitiescan be obtained with iohexol or iodixanol at a concentration of about10% to about 25% w/v. Sucrose can be used as a density cushion at aconcentration of about 10% to about 30% w/v e.g., about 15% to about 20%w/v, for blood culture samples. Other suitable materials that can beused to prepare the density cushion include low viscosity, high densityoils, such as microscope immersion oil (e.g., Type DF; Cargille Labs,New York), mineral oil (e.g., Drakeol® 5, Draketex 50, Peneteck®;Penreco Co., Pennsylvania), silicone oil (polydimethylsiloxane),fluorosilicone oil, silicone gel, metrizoate-Ficoll® (LymphoPrep™),e.g., at a concentration of about 75% to about 100% for blood culturesamples, diatrizoate-dextran (PolymorphoPrep™), e.g., at a concentrationof about 25% to about 50% for blood culture samples, carboxymethylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide (highmolecular weight), Pluronic® F127, Pluronic® F68, mixtures of Pluronic®compounds, polyacrylic acid, cross-linked polyvinyl alcohol,cross-linked polyvinyl pyrrolidine, PEG methyl ether methacrylate,pectin, agarose, xanthan, gellan, Phytagel®, sorbitol, Ficoll® (e.g.,Ficoll® 400 at a concentration of about 10% to about 15% for bloodculture samples), glycerol, dextran (e.g., at a concentration of about10% to about 15% for blood culture samples), glycogen, cesium chloride(e.g., at a concentration of about 15% to about 25% for blood culturesamples), perfluorocarbon fluids (e.g., perfluoro-n-octane),hydrofluorocarbon fluids (e.g., Vertrel XF), and the like as are wellknown in the art. In one embodiment, the density cushion is selectedfrom one or more of colloidal silica, iodixanol, iohexol, cesiumchloride, metrizoate-Ficoll®, diatrizoate-dextran, sucrose, Ficoll® 400,and/or dextran in any combination. The density cushion can also be madeup of a combination of materials, e.g., a combination of colloidalsilica and oil.

Transfer to Separation and Concentration Station (Centrifuge)

As shown in FIG. 21, after loading of the separation device 1904 withthe mixed or lysed test sample, the sample removal apparatus 1912retrieves the loaded separation device 1904, lifts it out of thecassette 1900, and moves the separation device 1904 to the centrifuge1916. The separator 1904 is then placed into a holder or loadingposition of the centrifuge 1916.

A separation and concentration of the microbial agent in the sampleoccurs within the separation device 1904 using the centrifuge 1916.

The separation step can be carried out to separate the microorganismsfrom other components of the sample (e.g., non-microorganisms orcomponents thereof) and to concentrate the microorganisms into a pelletthat can be interrogated for identification and characterizationpurposes. The separation does not have to be complete, i.e., it is notrequired that 100% separation occur. All that is required is that theseparation of the microorganisms from other components of the sample besufficient to permit interrogation of the microorganisms withoutsubstantial interference from the other components.

The centrifuge spins the separation device 1904 at high speed in orderto concentrate the microbial agent into the bottom of the capillary tubewithin the separation device 1904. The combination of the action of thelysis buffer on the non-microorganism cells (e.g., blood cells), thepresence of the density solution within the separation device 1904, andthe centrifugation, results in the separation of microbial agent fromthe lysed blood/broth mixture and the concentration of the microbialagent into a pellet or pellet-like mass in the bottom of the capillarytube, as shown in FIGS. 10 and 11.

In one embodiment, the separation device 1904 is centrifuged in station1916 using a swing out rotor so that the microorganisms form a pelletdirectly on the bottom of the separation device 1904 (in the bottom ofthe capillary tube shown in FIGS. 8, 10 and 13). The separation device1904 is centrifuged at a sufficient acceleration and for a sufficienttime for the microorganisms to be separated (e.g., a pellet formed) fromother components of the sample. The centrifugation acceleration can beabout 1,000×g to about 20,000×g, e.g., about 2,500×g to about 15,000×g,e.g., about 7,500×g to about 12,500×g, etc. The centrifugation time canbe about 30 seconds to about 30 minutes, e.g., about 1 minute to about15 minutes, e.g., about 1 minute to about 5 minutes.

Reading

The identification and/or characterization module (read station 1918),which is shown positioned adjacent to the centrifuge then interrogatesthe concentrated microbial agent using fluorescence spectroscopy (e.g.,intrinsic fluorescence and/or diffuse reflectance), Raman spectroscopyor other optical technique. In other embodiments, the microorganisms inthe pellet can be interrogated using mass spectrometry techniques, suchas MALDI-TOF mass spectrometry, desorption electrospray ionization(DESI) mass spectrometry, GC mass spectrometry, LC mass spectrometry,electrospray ionization (ESI) mass spectrometry and Selected Ion FlowTube (SIFT) spectrometry. As shown in FIG. 22, the identification and/orcharacterization module 1918 may be physically located proximate to thecentrifuge 1916, in which case the separation device 1904 does not needto be moved further by the robotic transfer mechanism. Alternatively,the identification and/or characterization module 1918 could be locatedin a different location within the identification/characterizationinstrument and the robotic transfer mechanism operates to move theseparation device to the location of the identification and/orcharacterization module 1918.

Transfer to Waste

After reading, as shown in FIG. 23, the robotic transfer mechanism 1910and sample removal apparatus 1912 operates to lift the separation device1904 from the centrifuge 1916, transfers the separation device 1904laterally and places it in the waste container 1908. FIG. 24 shows thewaste container 1904 containing a multitude of the sampling devices 1902and the separation devices 1908. When the waste container 1908 is fullit is removed from the instrument and then replaced with an empty wastecontainer. Prior to disposal of the separation device, a photographicimage of the lower region of the separation device may be taken with acamera (not shown) to verify the separation process and provide valuableinformation on the identity of the isolate, such as pellet size, shape,color and density.

External Processing of Concentrated Microbial Agent

While in the above embodiment the concentrated microbial agent isinterrogated while it is still located within the separation device1904, it is possible to remove the concentrated microbial agent from theseparation device and test it directly to identify and/or characterizethe microbial agent.

In this variation, referring to FIG. 25, the separation device 1904 istransferred to a removal device or station 4302. At the station 4302,the cap 2404 of the separation device 1904 is removed and theconcentrated microbial agent 2804 is removed from the separation device1904. The microbial agent is then subject to one or more additionaltests. In one possible configuration, the microbial agent is supplied toa molecular diagnostic test unit 4310 which may include a disposabletest strip or the like and processing instrument for identification ofthe agent. Alternatively, a sample of the microbial agent could beapplied to a MALDI mass spectrometry plate 4314 and the plate insertedinto a mass spectrometry unit 4312. Alternatively, the microbial agentcould be delivered to a microbial identification and/or characterizationtest device 4318 (e.g., test card) and the card incubated and tested ina processing instrument 4316.

III. Method of Operation

Flow chart (FIGS. 26A, B, C)

The method of operation of the identification/characterizationinstrument 104 in an embodiment in which the specimen container 500 issubject to both detection and identification steps will now be describedwith reference to FIGS. 26A-26C.

The process starts at step 4402 with the loading of a sample into one ofthe containers 500 and delivery of the loaded container 500 to adetection instrument (as described in our prior provisional applicationand in co-pending application Ser. No. ______, “Automated microbialdetection apparatus, attorney docket no. 01120). See FIG. 47, instrument102.

At step 4404, the container 500 is loaded into the detection instrument102, e.g., by placing the detection container on conveyer which deliversthe container to the detection instrument or by manually loading thecontainer. (See FIGS. 47 and 48 and the description of those figuresbelow)

At step 4406, the container 500 is incubated within the detectioninstrument 102.

At step 4408 the detection container is read (by a detection unit in theinstrument 102).

At step 4410, the reading of the detection container is analyzed todetermine if the container is positive. If no, the processing branchesalong NO branch 4411 and a check is made if a timer has expired (step4412). If the timer has expired, the bottle is deemed negative and thebottle is transferred to the waste container at step 4414. Otherwise,the incubation continues and steps 4406, 4408 and 4410 continueperiodically.

If at step 4410 the detection container is positive, the processingproceeds to the YES branch 4416. The detection container is moved to theexit location in the detection instrument at step 4418. At step 4420 thedetection container is transferred to theidentification/characterization instrument 104, e.g., by moving thedetection container 500 onto a conveyor and moving it into the entrancelocation of the identification/characterization instrument (see FIG.47). The transfer could occur by some other manner, the details of whichcan vary widely.

At step 4422 (FIG. 26B), the detection container is placed into one ofthe racks 2310 of the identification/characterization instrument 104.The robotic transfer mechanism 1910 may be used in this process.

At step 4424, the detection container is aseptically vented. This stepmay occur prior to picking up of the sampling device or may occur afterpicking up the sampling device, see FIGS. 15 and 16.

At step 4426, one of the sampling devices 1902 is picked up from thecassette 1900. The sampling device 1902 is pre-loaded with a selectivelysis buffer as shown in FIG. 15; alternatively the lysis buffer isadded to the sampling device at this time.

At step 4428, a protective cap (not shown), if fitted, covering theneedle 3202 of the sampling device is removed.

At step 4430, the needle 3202 is inserted into a upright ventedcontainer 500 (see FIGS. 16 and 17).

At step 4432, the detection container is inverted (see FIGS. 18 and 19)and a small sample (e.g., a 2.0 ml sample) is removed from the container500.

At step 4434, the container 500 is rotated to an upright orientation andthe needle 3202 of the sampling device 1902 is removed.

At step 4436, a small volume (e.g., 0.5 ml sample) of air is introducedinto the sampling device. This could be accomplished automatically usingthe pneumatic system 1914 connected to the sampling device.

At step 4438, a protective cap for the needle 3202, if fitted, isreplaced.

At step 4440, the sampling device 1902 is inverted and agitated tothoroughly mix the portion of the sample with the selective lysisbuffer.

At step 4442, the protective cap for the needle 3202, if fitted, isagain removed. (Note: a station fitted with appropriate gripping orgrasping features could be provided for automatically removing andreplacing the cap of the needle or alternatively the cap could remain onthe needle as described in the second embodiment)

At step 4444, a small portion of the positive broth/lysis buffer mix isdiscarded into a waste container.

At step 4446, the sample removal apparatus moves the sampling device1902 to the position above one of the separation devices 1904 (see FIG.38) and pierces the cap with the needle of the sampling device. Theseparation device 1904 is pre-loaded with the density cushion in thisembodiment.

In one possible variation, the lysis buffer is also loaded into theseparation device 1904 with the density cushion and the mixing of thesample and the lysis buffer takes place within the separation device1904.

At step 4448, the sample removal apparatus 1912 gently adds 0.5 to 1.0ml of the sample/lysis buffer mixture on top of the density cushionalready present in the reservoir of the separation device 1904. SeeFIGS. 20A and 20B.

At step 4450, the sample removal apparatus 1912 is moved to the positionof the waste container 1908 and the sampling device 1902 is discarded.See FIG. 20C.

At step 4452, the sample removal apparatus returns to the separationdevice 1904 and picks it up out of the cassette 1900 and moves it to thelocation of the separation and concentration station 1916, and placesthe separation device 1904 into the centrifuge. See FIG. 21.

At step 4454, the centrifuge cycle is started.

At step 4456, after completion of the centrifugation process, theseparation device is moved to the identification and/or characterizationmodule 1918 (reading station). Where the reading station is proximate tothe centrifuge, the centrifuge is rotated to a reading position whereinthe separation device 1904 is positioned for reading as shown in FIG.11.

At step 4458, the optical scan of the separation device 1904 in theidentification and/or characterization module is started (See FIG. 21,22).

At step 4460, after completion of the reading operation, the separationdevice 1904 is placed into the waste container 1908 (see FIGS. 23, 24).

IV. Second Embodiment FIGS. 27-46

A second embodiment of the identification instrument 104 will bedescribed in conjunction with FIGS. 27-46. This embodiment is similar tothe first embodiment of FIGS. 1-26 in terms of overall function andoperation; the main differences are (1) a different construction of therobotic transfer mechanism 1910; (2) a provision for vortexing of thesample and lysis buffer in the sampling device 1902, and (3) inclusionof optional detection features in the rack holding the specimencontainers 500 (see FIG. 28) for detecting microbial growth within thecontainer 500 so that the identification system is intimately combinedwith a detection system for detecting whether a specimen container ispositive for presence of a microbial agent. A few other points ofdifferentiation in the details of the configuration of the secondembodiment will also be noted in the following description.

However, the second embodiment, like the first embodiment of FIGS. 1-26,shares the same overall goals and design objectives. That is, the secondembodiment of FIGS. 27-46 automates the removal of a test sample from aspecimen container (preferably soon after a positive determination hasbeen made), automates lysing of non-microorganism cells in the testsample, automates loading of the lysed sample into a disposableseparation device, automates separation and concentration of themicrobial agent present in the lysed sample in the separation device,and automates interrogation of the microbial agent to identify and/orcharacterize the microbial agent.

The culture bottles/specimen containers 500 are loaded into racks orholding structures of the identification instrument 104 either manuallyor automatically. In an optional configuration, the specimen containers500 are tested for the presence or microorganisms by a detectionsubsystem which is incorporated into the racks. In a manual, prior artmethod, without automation, a technician would remove a bottle from aseparate detection instrument after the bottle is deemed “positive”.This could be several hours after the diagnostic determination,especially if the determination is made in the middle of the night orwhen the lab is understaffed. However, with the automated identificationinstrument in this embodiment, the steps of automated identificationand/or characterization of the microbial agent can proceed immediately,and automatically, after the specimen container is deemed “positive”.

In the case of lytic centrifugation and intrinsic fluorescencemeasurement, features of both of the illustrated embodiments, it may bedesirable that the sample be processed for purposes of identificationand/or characterization shortly after a positive call by an associateddetection instrument. As the bottle is called positive themicroorganisms are in an exponential stage of growth. This growth phaseis distinguished from the lag phase and death phase which are bothbefore and after, respectively, the exponential phase. Microorganisms inthis exponential phase have different physical and genetic expressioncharacteristics than the lag and death phase.

By automating this process of identification and/or characterization,the technician is removed from the system. Identification and/orcharacterization of the microbial agent can occur much more rapidly inthe present embodiments as compared to prior approaches.

A. System Layout

The identification instrument 104 in accordance with a second embodimentis shown in FIG. 27. The instrument 104 includes a first cassette 1900Acontaining a plurality of disposable sampling devices 1902 and a secondcassette 1900B containing a plurality of disposable separation devices1904. A rack or holding structure 1906 includes receptacles for holdinga multitude of containers 500 containing samples for identificationtesting. The rack 1906 is shown contained within an insulated incubationenclosure 1812. The enclosure 1812 includes a door 1810 that is openedto expose the bottles 500 and allow venting of the bottles and removalof a test sample via a robotic transfer mechanism 1910, sample removalapparatus 1912, and the sampling device 1902.

The robot transfer mechanism 1910 includes a rotating base and movablejoints and segments between the joints so as to allow the robotictransfer mechanism 1910 and in particular gripping structures includedin the sample removal apparatus or sampling head 1912 to access thevarious components in the instrument 104. These components include aseparation and concentration device (centrifuge 1916), the cassettes1900A and 1900B, a vortexer 1814 for mixing a lysis buffer and testsample within the sampling device 1902, a read station 1918, and variouscontainers 1802, 1804, 1806 containing different lysis buffers and/ordensity cushions in the situation where the lysis buffers and densitycushions are added to the sampling device or separation device at thetime of use. The robotic transfer mechanism 1910 is able to access eachof the bottles 500 and optionally grip and hold the bottles 500. Thus,the robotic transfer mechanism 1910 may optionally be the device toautomatically load the bottles 500 into the holding structure or rack1906. Alternatively, the bottles 500 could be loaded into the rackmanually via an access door positioned on the opposite side of theenclosure 1812 from the door 1810. See FIG. 49, door 4902.

In the configuration of FIG. 27, a centrifuge cup holder 1800 holds asmall cup-like holder 1801 into which the separation device 1904 isplaced (see FIG. 46A); the combination of the separation device 1904 andcup-like holder 1801 are placed into the centrifuge 1916 for separationand concentration of the microbial agent in the separation device 1904.After centrifugation, the cup-like device 1801 is returned to the cupholder 1800. The sample removal apparatus 1912 grips the separationdevice and the robotic transfer mechanism places it into thereading/identification module 1918. The concentrated microbial agent inthe separation device 1904 is interrogated by the reading/identificationmodule 1918. The reading step may include the features described above,such as measuring intrinsic fluorescence spectra of the sample and, withthe aid of a computer, comparison of the measured spectra to a data setcontaining spectra from known microbial agents and classification usinga classification algorithm. After reading, the separation device 1904 isplaced into a waste container (see FIGS. 1, 15, item 1908).

FIG. 28 is an illustration of an alternative arrangement for theidentification instrument 104. In this embodiment, the walls or panelsfrom the incubation enclosure are removed to show one embodiment of theracks 1906 that hold the bottles 500. The racks 1906 are incorporatedinto a rotating turret which rotates about a vertical axis. Detectioninstrumentation for noninvasively detecting whether a bottle is positiveis incorporated in the racks 1906. These aspects are described in moredetail in co-pending application Ser. No. ______, attorney docket no.01145/MBHB 09-271-E, filed on the same date as this application, thecontent of which is incorporated by reference herein. Accordingly, inthis embodiment the racks 1906 and associated detection instrumentationfunction as an automated detection system 102 for determining thepresence of a microbial agent in a specimen container.

FIG. 29 is another perspective view of the embodiment of FIG. 27,showing the centrifuge 1916, vortexer 1814 and sample removal apparatus1912 included in the robotic transfer mechanism 1910.

FIG. 30 shows the cassettes 1900A and 1900B of disposables in moredetail. While each cassette is shown holding twenty-five disposablesampling devices 1902 or separation devices 1904, the number orarrangement of the devices 1902 and 1904 within a replaceable cassette1900 is not important.

B. Robot Transfer Mechanism 1910 and Sampling Removal Apparatus 1912

FIG. 31 is a perspective view of the robotic transfer mechanism 1910.The transfer mechanism 1910 is shown in a form of a six-axis robot. Therobot includes six rotational joints 1700 indicated by the arrows andsegments 1702 between the robot joints that expand or contract linearlyin order to extend or contract or otherwise move the position of thesample removal apparatus 1912 placed at the tooling end of the robot armin three-dimensional space. A rolling diaphragm pump assembly 1710 isfitted to the robot transfer mechanism 1910 to apply vacuum or positivepressure to the sampling device 1902 via a connecting tube 3402 tofacilitate venting and sampling the containers 500 as described below.The pneumatic system 1914 (FIG. 1) provides pneumatic controls for thegripping tooling forming the sample removal apparatus 1912.

A six axes robot is chosen to allow for flexibility especially theability to vent and sample the bottle. A pneumatic gripper 1954, seeFIGS. 34 and 35, is placed at the tooling end of the robot on the lastrotary joint. Plates attached to the gripper 1954 hold three endeffectors; that is, there are three separate gripping components: one1956 for the sampling device 1902, one 1958 for the separation device1904 and specimen containers 500, and one (not shown) for a vacuum tubewhich may be used in future configurations. A connector 1952 is used toattach the free end of the tube 3402 to the fitting 3208 (FIG. 33) onthe proximal end of the sampling device 1902. A pneumatically operatedlinear slide 1950 is moved forward to advance the connector 1952 toengage with the sampling device 1902 and backward to disengage from thesampling device when the device 1902 is deposited in the wastecontainer.

The gripper 1954 and linear slide 1950 are pneumatic driven, with thegripper and linear slide controlled from the same air line (3602 FIG.36). Flow control valves (not shown) control the rate movement on thegripper and linear slide. When the sampling device 1902 is to be pickedup, the gripping component 1956 is positioned around the sampling device1902 with the component 1956 open and the linear slide 1950 refracted.An air valve (not shown) is activated to close the gripper 1954 andclose the gripping component 1956 and advance the linear slide 1950.Through flow controls, the gripper closes first, grabbing the samplingdevice 1902. Shortly after, the linear slide 1950 advances and engagesthe connector (1952 in FIG. 34) with the sampling device 1902. Tubing3402 connects the pump assembly 1710 with the connector 1952 in FIG. 34and sampling device 1902, thereby establishing a connection from thesampling device 1902 to the pump 1710 (FIG. 36) via the tubing 3402.

C. Sampling Device 1902

The sampling device 1902 in this embodiment is shown in FIGS. 32 and 33.The operation of the device 1902 for venting and sampling a specimencontainer 500 is described below in conjunction with FIGS. 36 and 37.The operation of the device 1902 for injecting the sample into theseparation device 1904 is described below in conjunction with FIGS.43-46.

Referring now to FIGS. 32 and 33, the sampling device 1902 includes a18-gauge needle 3202 having a rubber sheath 3214. A luer fitting 3212connects the needle 3202 to a 5 ml syringe body or tube 3200. A 0.2 μmhydrophobic filter 3218 is coupled to the syringe body 3200 with anelastomeric fitting 3210. A port or fitting 3208 receives the tube 3402(FIG. 34) which is connected to the rolling diaphragm pump 1710 fittedon the robot transfer mechanism 1910 of FIG. 31.

The sheath 3214 has four functions: 1) sheath the needle 3202 to avoidneedle stick injuries, 2) keep needle 3202 sterile, 3) prevent leakingof components out of tube 3200, and 4) act as spring to push back oncomponents during sampling from the specimen container 500 and theinjection of the separation device 1904 (see FIGS. 44 and 45). Thesheath prevents the needle 3202 from sticking or binding to a septum orstopper fitted on the end of the specimen container 500. As the needleis withdrawn from the septum the rubber sheath pushes against the septumpreventing the binding of the needle and septum. Similarly, duringinjection of the separation device the spring-like compression of thesheath 3214 pushes against the screw cap of the separation device 1904and prevents the needle for sticking or binding to the cap.

The hydrophobic filter 3218 (FIG. 33) prevents microbes fromcontaminating the pumping system and tubing. Since this filter ishydrophobic, liquid is prevented from passing to the pump 1710 of FIG.31. Another function of the filter besides preventing contamination, isrepeatable fluid withdrawal. Once liquid touches the filter 3218 no moreair can be evacuated from the tube 3200 since the water blocks the flowof air. Thus, the pump 1710 can continue pumping but the volume ofliquid extracted will be a function of the tubing volume and not theprecision of the pump.

D. Vacuum Pump Assembly 1710

The vacuum pump assembly 1710 of FIG. 31 is shown isolated inperspective view in FIG. 36. The pump 1710 contains a rolling diaphragm1712 connected to a linear actuator 1714. Solenoid valves 1716 and 1718switch the pump from input to output. During the venting step, in whichthe bottles 500 are vented to atmosphere using the sampling device 1902,the solenoid valves 1716 and 1718 are actuated to let positive pressurevent through the system (input). Also, during sampling the pump draws influid from the bottle 500 into the sampling device 1902. The fluid isejected (output) to the separator device 1904 (FIGS. 43-44). For bothinput and output modes, the linear actuator 1714 continues to operate,moving the rolling diaphragm 1712 back and forth. Check valves (notshown in FIG. 36) take part in controlling the direction of the fluid.

E. Venting and Sampling

The venting and sampling steps are shown in FIGS. 37 and 38. The robot1910 first picks up one of the venting and/or sampling devices 1902 fromthe cassette 1900A. The robot 1910 moves into the position shown in FIG.37. The door 1810 is opened. The racks 1816 of FIG. 37 are rotated to anupwards pointing position and venting occurs by means of inserting theneedle of the sampling device 1902 into the specimen containers 500. Theracks 1816 are then rotated to a downward pointing position shown inFIG. 37 and the pump 1710 operated to draw a small volume of the sample(0.5 to 1.0 ml) from the specimen container 500 into the sampling device1902. The sampling device 1902 is either pre-loaded with lytic agent oralternatively the lytic agent is added to the sampling 1902 prior to theventing and sampling steps. In the case where the sampling device isloaded with lytic agent in-situ, the robotic transfer mechanism graspsone of the sampling devices, accesses lytic agent solutions stored incontainers 1802, 1804, 1806 etc., see FIG. 27, and withdraws 1.0 to 2.0ml of lytic agent into the sampling device 1902 and then proceeds withthe venting and sampling steps.

F. Mixing of Lytic agent and Sample in Sampling Device 1902

As noted previously, the embodiment of FIGS. 27-46 includes features formixing the test sample and lysis buffer, e.g., by vortexing the samplingdevice 1902 in order to mix the sample withdrawn from the specimencontainer with the lytic agent present in the sampling device 1902.

The vortexing will now be described in conjunction with FIGS. 29, and39-42, showing the vortexer 1814. A unique feature of this system is avortex cup 3900 which holds the sampling device 1902. The robotictransfer mechanism 1910 first places the sampling device 1902 in thevortex cup 3900 (see FIG. 39), releases the sampling device 1902, andthen moves upward into the position shown in FIG. 40. The robot gripperfingers 3450 then closes loosely around the above the hydrophobic filter3218 (FIGS. 32, 33) of the sampling device 1902. The sampling device1902 is held loosely in the vortexer cup 3900 so that the vortexer 1814can be free to agitate the sampling device 1902. If it is held tightlythe vortexer 1814 is not free to agitate the sample/lysis buffer mixturewhich is present in the sampling device 1902. The bottom surface 1957 ofthe gripper tooling 1956 retains the sampling device 1902 in thevortexer 1814 during vortexing.

The vortexer 1814 includes a base 3902 that the cup or holder 3900 ismounted to as via fasteners extending through holes 4202 in the flange4204 of the holder 3900 as shown in FIGS. 41-42. The interior channel4200 of the holder 3900 is sized to fit the sampling device 1902 asshown in FIGS. 40 and 41. The vortexing sufficiently mixes the sampleand the lysis buffer with a 5 second cycle at 3000 rpm.

In one optional configuration, the vortex cup 3900 include heatingelements to maintain the sample in the sampling device 1902 at 37degrees C. The heating may take the form of a coil resistive heater 3910shown in FIG. 39A. The agitation frequency, duration and temperature ofthe vortex process may change for particular samples and buffers.

G. Injection of Mixed Sample into Separation Device 1904

It may be desirable to first load the separation device into thecentrifuge to pre-spin the lytic buffer and insure no trapped air ispresent in the capillary tube of the separation device. Also, if theoptics system is configured in the centrifuge a quality check (e.g., apre-read of the separation device before adding lysed sample) can beperformed. Quality control checks could include inspection for debris orfibers that may be present in the separation device, scratches to theoptic surfaces, or other optic defects.

After the vortexer 1814 completes the mixing of the sample and lysisbuffer in the sampling device 1902, an approximately 1 ml portion of themixed sample and lysis solution is then injected into the disposableseparation device 1904. This operation may occur while the separationdevice 1904 is still contained within the cassette 1900B of FIGS. 26 and27. The mixed sample and lysis buffer is shown as mixture 4302 in FIG.43A. (FIG. 43D shows the rubber sheath 3214 shown partially removed fromthe needle 3202 but this is only to better illustrate the sheath and theneedle; the needle is covered by the sheath during use as shown in FIGS.43B and 43C).

To accomplish the injection of the sample into the separation device1902, the robotic transfer mechanism positions the (loaded) samplingdevice 1902 over one of the separation devices 1904 as shown in FIG. 43Aand then proceeds to lower the sampling device 1902 so that the needle3202 is forced through both the rubber sheath 3214 and the septum 4300provided in the cap 2404 of the separation device 1904 so as to placethe tip of the needle 3202 into the interior chamber 2602 of theseparation device. See FIGS. 44, 45 and 46. This action compresses therubber sheath 3214 as shown in FIGS. 44, 45 and 46, with the sheath 3214acting on a spring and applying force to the cap 2404. As shown in FIG.46, the roller diaphragm pump 1710 operates to pump air into thesampling device 1902, creating positive pressure in the interior of thesampling device and thereby forcing the test sample/lysis buffer mixture4302 to be injected via the needle into the separation device 1904 asshown in FIG. 46. The mixture 4302 is dispensed on top of the 1.0 mldensity cushion 2802 already present in the separation device 1904.

H. Transfer of Loaded Separation Device 1904 into Centrifuge 1916

After loading of the separation device 3202 in this manner, the robotictransfer mechanism 1910 proceeds to transfer the sampling device 1902 toa waste container, and then pick up the loaded separation device 1904and place it in the cup 1801 held by the cup holder 1800 (FIG. 28, FIG.46A-46C). Then, the combination of the cup 1801 and separation device1904 is picked up and lifted off the holder 1800 (FIG. 46A) by therobotic transfer mechanism 1910 and placed in the centrifuge 1916 (FIG.28) for separation and concentration of the sample in the separationdevice 1904.

In one possible embodiment, the centrifuge 1916 is not an indexedcentrifuge, i.e., it does not come to the exact same position afterspinning. The centrifuge lid is open and closed by a pneumatic cylinder.The position of the centrifuge is found by a camera (not shown) on therobot transfer mechanism 1910. A picture of the centrifuge is taken andmachine vision software determines the position of the centrifuge sothat the separation device 1902 can be correctly placed in thecentrifuge. In particular, the camera looks for a fiduciary mark on therotor and the robot moves to the appropriate position in the centrifugerotor. The separation device 1904 is inserted into the proper locationto maintain balance in the centrifuge 1916.

The centrifuge could be configured to just spin one separation device ata time (as in the case of the first embodiment), or multiple devices ata time as shown in FIGS. 27-29.

The machine vision component (camera) could be eliminated by using anindexed centrifuge rotor. In this configuration, the centrifuge rotorwould stop at the same position after centrifugation. This could beaccomplished by using a mechanical clutch to engage the rotor and movingit past an optical sensor to the correct position. This method couldeliminate complexities (e.g. lighting, complex software algorithms) andcosts associated with machine vision, and thus for some implementationsmay be preferred.

I. Separation and Concentration of Microbial Agent in Separation Device1904

The centrifuge operates to spin the separation device 1902 at highrevolutions per minute for sufficient time to concentrate themicrobiological specimen within the separation device into a pellet orpellet-like mass, as described in conjunction with the first embodiment,e.g., 10,000 g for 2 minutes. During centrifugation the lysed red bloodcells separate to the top of the density cushion and the intact microbesform a pellet at the bottom of the 1 mm capillary tube 2604 in theseparation device 1902 (see FIG. 43A). The centrifuge lid is openedusing a pneumatic cylinder and the robot removes the separation device1902 and cup 1801. The position of the capillary tube and holder isdetermined by machine vision as in the placement step above. Theseparation device 1902 and cup 1801 are removed as a unit from thecentrifuge 1918 and placed on the cup holder 1800 (using pin 1805 as alocating mechanism, see FIG. 46C), and then the robot 1910 picks up theseparation device 1902 and moves it to the reading unit 1918.

J. Reading of Concentrated Microbial Agent in Separation Device 1904

The reading unit 1918 interrogates the concentrated microbial agentforming the pellet within the separation device 1902. The details arenot pertinent to this disclosure. The results (characterization and/oridentification information for the microbial agent) are output to theuser interface of the instrument, a connected workstation, a printer, orother output device depending on the configuration of the instrument.

K. Sterilization of Specimen Container 500 Stopper

In some biological applications for the present instrument 104, thespecimen container 500 is inoculated with a specimen sample, such ashuman body fluids or other normally-sterile body fluids. This isaccomplish by injecting the sample through a needle into a stopperformed at the top of the container 500. There is a chance that thesample may contain biohazardous material. Often a small drop of thesample, such as blood, may be left on the surface of the stopper. It isdesirable to sterilize this surface before sampling and processing toavoid contamination of the container 500 with airborne or surfacemicrobes.

Several methods could be developed to sterilize this surface in anautomated manner. These include:

1) UV sterilization of the stopper surface. Ultraviolet light is astandard method of sterilizing surfaces. Automation could beaccomplished by attaching a UV light source to a second robot orautomation mechanism provided in the instrument that would move to thestopper surface for sterilization before venting the bottle or removinga test sample.

2) Misting the surface with a disinfectant such as isopropyl alcohol orother chemical and then wiping the surface clear. Presently this is themost common manual method of sterilizing inoculation sites. Normally,swabs are soaked in a disinfectant and a technician wipes the surfacebefore inoculating the bottle or removing a sample. Mechanical wiping isnecessary in the case of dried blood spots on the surface since achemical mist may not penetrate through the blood. The misting of thesurface can be automated by pressurizing a disinfectant reservoir withair and spraying this onto the surface of the stopper. The mechanicalwipe can be accomplished by picking up a swab or fabric wipe and wipingthe stopper surface. Other mechanical methods of wiping the surfaceinclude a rolling fabric soaked in the disinfectant. Again, thesemethods could be accomplished by means of a separate robotic mechanismin the instrument 104, or by providing the existing robot transfermechanism 1910 with additional gripping/wiping/misting/UV sterilizationcomponents as the case may be.

L. Other Configurations for Robotic Transfer Mechanism 1910

While the second embodiment shown in FIGS. 27-29 uses a six-axes robotfor the automation robot transfer mechanism 1910 to accomplish transferand positioning of components or materials in the instrument, it is butone of a variety of choices that could be made and the scope of thepresent disclosure is intended to encompass other robotic transfermechanisms. A multi-axis robot arm was chosen because it is flexible.New automation steps can be easily programmed without requiring majormechanical tooling redesigns. Once the process is established, the robotcould be replaced by a simpler and more compact robot, with fewer axes,or a Cartesian (x,y,z) automation system. The Cartesian system would bemore inexpensive than the six-axes robot. A Cartesian system is used forexample in the first embodiment (see FIG. 5).

M. Electric Actuators

A few of the actuators of the second embodiment (and in particular thegripper and slide aspects of the sample removal apparatus 1912) areoperated by pneumatics (compressed air). Pneumatic mechanisms are simpleto program and design, however they are not amenable to clinical or somelaboratory settings where compressed air is not available. Theseactuators can be replaced by electrical/mechanical systems such aslinear drives, stepper and servo motor connected to linear drives andsolenoids.

N. Alternative Mixing Methods

In the second embodiment, a vortexer 1814 is used to vigorously mix thesample and lytic buffer. A different mixing method such as sonication orreciprocal mixing could be used in place of vortexing.

O. Other Applications for Identification System

We have described in detail a method and instrument for automaticallyvent and sample a specimen container, e.g., blood culture bottle. Thesample is lysed and centrifuged to process the microbial agent presentin the sample for further analysis. The features of the instrument canbe applicable to other diagnostic systems and other types of culturebottles. These systems could include molecular biology tests orautomated culture bottles for industrial samples. Industrial samplescould include sterility testing of drugs or food.

In the case of molecular biology tests it may be very important toperform a microbial test during exponential growth of a microorganism.During the exponential growth phase the genetic expression of microbesis different than during the lag phase. In the lag phase, which is priorto the exponential growth phase, microbes are converting their geneticmachinery to express proteins to consume the media nutrients which maybe different from their previous environment. As the microbes enterexponential phase the genetic expression has become set.

An automated detection instrument (102), such as that described here andin our prior provisional application or the BacT/ALERT® system, candetermine when the microbes begin exponential phase and the automatedidentification method above can process a sample soon after exponentialphase begins. In a manual culture method it would be difficult todetermine when exactly the microbes enter into exponential phase sincethis would entail checking the bottles frequently for turbidity. Shouldthe beginning of the exponential phase be missed by the technician,there is a risk that microbes would pass into death phase as the limitednutrients are consumed. Hence, in preferred embodiments the presentidentification instrument automatically processes the positive specimencontainers soon or immediately after the container is deemed “positive.”

In some other non-clinical embodiments of the identification system, thelysis step is optional or not preformed. Hence, the provision of a lyticbuffer in the sampling device and vortexing the sampling device are notrequired in every possible configuration of the present inventiveinstrument.

P. Re-Sampling of Specimen Containers

The process of venting, sampling, separation and interrogation describedabove can be repeated on the same specimen container 500 as needed. Inone possible variation, a given specimen container 500 is sampledsuccessively using sampling devices 1902 loaded with different lyticbuffers (e.g., loaded in situ from the supply of lytic buffers in theinstrument) and loaded into different separation devices 1904 which arethen subject to separation and concentration steps and then reading.

The instrument 104 may also perform identification and/orcharacterization testing without first performing the detection step;possibly shortening the time to identification. This mode of operationcould be employed when other clinical data are available that arepredictive of infection. Patient condition, biomarkers (e.g., PCT) etc.are examples of data that could be predictive of infection. In thismode, specimen containers are loaded into the identification instrument104 (e.g., using the rack designs of either embodiment), the bottles areincubated in racks provided in the identification instrument, and everybottle is periodically sampled and subject to the separation andconcentration step and the interrogation step. If a given sample is notable to be identified or characterized at the first iteration, thespecimen container can be re-sampled, e.g., every 30 minutes, untilsufficient microbial agent growth has occurred within the specimencontainer such that the reading step for that subsequent iterationreturns an identification and/or characterization result. Incubation ofthe specimen container occurs prior to and during the sequentialsampling of the specimen container.

Q. Coupling to Automated Detection Instrument.

In some embodiments, the automated identification instrument 104 of thefirst and second embodiments is tightly coupled to an automateddetection instrument configured to determine whether a specimencontainer 500 is positive for presence of a microbial agent. This tightcoupling preferably provides for automated hand-off of positive specimencontainers 500 from a detection instrument to the automatedidentification instrument 104 as soon as the specimen container istested “positive.”

A variety of instrument configurations for achieving such coupling aredescribed in our prior U.S. provisional application Ser. 61/216,339filed May 15, 2009. A few options are shown in FIGS. 47 and 48. In FIG.47, an automated detection instrument 102 is linked via conveyer 4702 tothe automated identification and/or characterization instrument 104.Bottles arriving at the automated identification and/or characterizationinstrument 104 are picked up by the robotic transfer mechanism 1910 andloaded into the racks. In FIG. 48, the bottles are provided to acombined detection and automated identification and characterizationinstrument (e.g., as set forth above for the second embodiment, see FIG.28 and the above discussion). In this configuration, the racks holdingthe incoming specimen containers 500 include detection instrumentationfor interrogating colorimetric sensors incorporated in the bottom of thebottles. Further, the combined instrument 102+104 is provided withincubation features, such as providing the incubation enclosure 1812 ofFIGS. 27 and 37.

Still other configurations are possible, as describe in the co-pendingapplication Ser. No. ______ filed on the same date as this application,attorney docket no. 09-271-US. FIG. 49 shows an embodiment in which thecombined instrument 102+104 includes a door 4902 for manual loading ofbottles into the racks of the combined detection andidentification/characterization instrument.

In the embodiments of FIG. 47-49, a drawer 4702 is provided to provideaccess to remove waste from the instrument, e.g., specimen containers,sampling devices and separation devices.

The physical configuration of the external panels for the instruments ofFIGS. 47-49 are not particularly important and can vary widely. Theinstruments include a graphical user interface and display 4704 whichcan also vary widely.

R. Computer System Schematic

FIG. 50 is schematic block diagram showing the identification and/orcharacterization instrument 104 and its associated computer controlsystem. The details shown in FIG. 50 can vary widely and are notparticularly important, and therefore are provided here by way ofexample and not limitation.

A computer 4902 running LabVIEW (National Instruments) is connected totwo computers: (1) a computer 4904 via a serial connection, and (2) arobot control computer 4906 via an Ethernet connection. The computer4904 controls the racks 1906 and associated detection subsystem fordetecting whether bottles are positive, controls the stepper motorswhich agitates (oscillates) the rack 1906 to provide agitation duringincubation via a motion controller 4908. A stepper motor (not shown)allows for the rack to be precisely put in position for venting andsampling by the robot transfer mechanism 1910.

The LabVIEW 4902 computer queries the computer 4904 for positivebottles. The computer 4904 computer replies through the serialconnection and the bottle ID, time of positive and bottle position areparsed by the LabVIEW computer 4902. The bottle position is sent to therobot controller 4906 which opens the door to the racks (FIG. 27, 1810)through a digital signal to a relay controlling pneumatic cylindersconnected to the door. The robot 1910 acquires a sampling device 1902and vents the bottle and samples as described above.

A digital signal from the robot controller 4906 is sent to relays toopen and close the lid of the centrifuge 1916, start the centrifuge 1916and control the vortexer 1816. Motion control of the linear actuator onthe rolling diaphragm pump is controlled by the LabVIEW computer 4902via a motion controller 4908.

Interrogation measurements (e.g., intinsic fluorescence measurements)aptured by the identification module 1918 are sent to the LabVIEWcomputer 4902. The computer 4902 compares the measured spectra withstored reference spectra from known samples to identify and/orcharacterize the microbial agent in the sample as described above. To dothis comparison, the computer 4902 includes a memory (e.g., hard disk)containing the reference spectra data and machine-readable code storingsoftware instructions to perform the comparison, e.g., the algorithmsdescribed previously. The computer 4902 includes a conventional centralprocessing unit which operates on the acquired data and stored referencedata using the algorithm(s) to generate a result for the sample undertest and provides a report e.g., via a user interface on the instrumentor attached peripherals 4910. The computer 4902 can communicate over anInternet Protocol network 4914 with other remotely located computers4912, e.g., to share identification and/or characterization results,store the results in a remote database, or interface to other laboratoryinformation systems.

S. Combination of Separation and Sampling Devices into a SingleDisposable Device.

As described previously, the identification and/or characterizationinstrument 104 includes a sample removal apparatus 1912 which holds orgrasps a disposable venting and/or sampling device 1902. Together, theyoperate to vent the specimen container and remove a portion of thebiological sample in the positive detection container 500 and add theportion to a separation device 1904. The functions of vending andsampling could be performed by separate disposable devices 1902.Additionally, the functions of venting, sampling and separation could beperformed in a single disposable device as described in this section.

Referring to FIGS. 51-55, a separation device 6000 includes a body 6002,generally in the shape of a block, a top plate 6004 and a base plate6006. The body contains an optical window used for intrinsicfluorescence measurements; the material forming the window opticallyclear and non-fluorescing. In general, the body 6002 can be molded orotherwise formed from any known plastic material known in the art. Asshown in FIGS. 53-55, the body 6002 of the separation device 6000encloses a lytic chamber 6020, a venting channel 6030, a fluid transferchannel 6034 and a separation chamber 6040. The lytic chamber 6020 andseparation chamber 6040 are orientated along two parallel and adjacentvertical axes 6022, 6042, defined in the body 6002, each chamber havingtop and bottom terminal ends. The venting channel 6030 provides a firstfluid communication channel connecting the bottom end of the lyticchamber to a vent or pump port 6018 in the top plate 6004. As shown inFIGS. 54-55, the first fluid communication channel further comprises aventing fluid flow groove 6032 contained in the upper surface 6034 ofthe body 6002 and providing fluid communication between the lyticchamber 6020 and the venting channel 6030. The fluid transfer channel6036 provides a second fluid communication channel connecting the bottomend of the lytic chamber 6020 to the top end of the separation chamber6040 for transferring a lytic buffer and sample from the lytic chamber6020 to the separation chamber 6040. As shown in FIGS. 54 and 56, thesecond fluid communication channel further comprises a venting fluidflow groove 6038 contained in the upper surface 6034 of the body 6002and providing fluid communication between the lytic chamber 6020 and theseparation chamber 6040. The lytic chamber 6020, venting channel 6030and fluid transfer channel 6036 are open to a bottom surface 6010 of thebody 6002 of the device 6000, as shown in FIG. 52. The bottom surface6010 of the body 6002 may further comprise a lower fluid flow groove6024 providing fluid communication between the bottom of the lyticchamber 6020 and the venting channel 6030 and fluid transfer channel6036 through a valve well 6026 (described further below). The top plate6004 and base plate 6006 can be attached to the body 6002 by any knownmeans in the art to close of or otherwise seal the chambers 6020, 6040and channels 6030, 6034. For example, the top plate 6004 and/or baseplate 6006 can be affixed to the body by welding or by the use of anadhesive.

As shown in FIG. 52, the separation device 6000 includes a valve 6012and a valve actuator port 6008 that runs through the top plate 6004. Thevalve 6012 is contained within a valve well 6026 in the bottom surface6010 of the body 6002, and is operable between a first position and asecond position via an external actuator (not shown). When the valve6012 is in a first position, a first fluid communication channel is“open” from the bottom of the lytic chamber 6020 through the ventingchannel 6030 to the vent or pump port 6018. This open first fluidcommunication channel is operable to vent excess pressure from thedevice 6000 or to provide a vacuum to the lytic chamber 6020 through theuse of a pump (not shown). When the valve 6012 is in a second position,a second fluid communication channel is “open” from the bottom of thelytic chamber 6020 through the fluid transfer channel 6036 to theseparation chamber 6040. This open second fluid communication channel isoperable for transferring the lytic buffer and sample from the lyticchamber 6020 to the separation chamber 6040. As shown in FIG. 62, thevent or pump port 6018 and sample entry port 6016 comprise open channelsthrough the top plate 6004. In one possible embodiment, the sample entryport 6016 further comprises a pierceable septum (not shown). In anotherembodiment, a syringe needle (not shown) can be attached or affixed tothe sample entry port 6016, thereby allowing the lytic and separationdevice to additionally operate as a venting and/or sampling device fordirectly obtaining a sample from a specimen container 500.

As shown in FIG. 54, the separation chamber 6040 may further comprise anupper reservoir, a middle tapered section and a lower capillary tube6048 all arranged around a central vertical axis. As shown, the middletapered section connects the wider diameter upper reservoir and thesmaller diameter capillary tube 6048. In one embodiment, the bottom wallof the capillary tube 6048 is made of an optically transparent materialfor facilitating optical interrogation of a concentrated microbial agent(not shown) located at the bottom of the capillary tube 6048. In anotherembodiment, the separation device 6000 is made of an opticallytransparent material to facilitate optical interrogation of aconcentrated microbial agent (not shown) located at the bottom of thecapillary tube 6048. As shown, the bottom wall opposite the capillarytube 6048 may be of a reduced thickness to facilitate opticalinterrogation as indicated in FIG. 53. In yet another embodiment,optical interrogation can occur from the side of the device 6000. Inaccordance with this embodiment, the block will comprise a notch section6010 and a reduced thickness side wall juxtaposed the capillary tube6048. In accordance with this embodiment, the separation device 6000 ismade of an optically transparent material to facilitate opticalinterrogation of a concentrated microbial agent (not shown) located atthe bottom of the capillary tube 6048.

In operation, the lytic chamber 6020 can be loaded with a lysis bufferand a sample taken from a positive culture container. For example, asampling device 1902, as described elsewhere herein, can be used todeposit separately or in combination a lysis buffer and a sample from apositive culture container into the lytic chamber 6020. In anotherembodiment, the lysis buffer can be added to the lytic chamber 6020 ofthe separation device 6000 within the characterization/identificationsubsystem. For example, the sampling device 1902 can be used to obtainan aliquot of lysis buffer (e.g., from a lysis buffer reservoir) thatcan be subsequently deposited into the lytic chamber 6020 through thesample entry port 6016 (e.g., a pierceable septum) in the body 6002.Next, the sampling device 1902 can be used to obtain a sample from apositive specimen container 500 and deposit that sample into the lyticchamber 6020 through the lytic chamber port 6016. The lysis buffer andsample are then mixed within the lytic chamber 6020, e.g., by agitationand/or vortexing of the sampling device 6000. The selective lysis stepis allowed to proceed for a sufficient time to allow the lysis reactionto be substantially completed (e.g., from 1 to 5 minutes). Thisselective lysis step selectively lyses undesired cells (i.e.,non-microorganism cells) that may be present in the sample, e.g., bloodcells and/or tissue cells. In another embodiment, the lytic chamber 6020can be pre-loaded with a lysis buffer and the sample loaded to the lyticchamber prior to agitation and/or vortexing. In one embodiment, thesampling device 6000 can optionally be incubated to allow the selectivelysis step to proceed more quickly.

After the lysis step, the lysed sample and lysis buffer can betransferred to the separation chamber 6040 through the a fluid flowchannel 6030 for the separation of any microorganisms over a pre-loadeda density cushion, as described herein. The valve 6012 is pressed downexternally by a mechanical actuator (not shown), thereby opening thefluid flow channel 6030 between the lytic chamber 6020 and theseparation chamber 6040. A pump above the separation chamber 6040 drawsthe mixture through the fluid flow channel 6030 to the top of theseparation chamber 6040. In one embodiment, by holding the separationdevice 6000 at an angle, the fluid can flow gently down the interiorwall of the separation chamber 6040 and onto the density gradient.

The identification/characterization instrument 104 further includes aseparation and/or concentration station, optionally in the form of acentrifuge, which operates on the separation device 6000 so as toseparate the microbial agent from other products in the portion of thebiological sample and concentrate the microbial agent within theseparation device 6000. In one example, the microbial agent isconcentrated in the form of a pellet or pellet-like mass in the bottomof the capillary tube 6060 of the separation device 6000.

The identification/characterization instrument further includes aidentification and/or characterization module or read station (see,e.g., FIG. 1, 1918) which interrogates the concentrated microbial agentto identify and/or characterize the microbial agent.

Another embodiment having a stacked chamber design is shown in FIGS.59-69B.

FIGS. 55-60 illustrate a combined sampling and separation device 6100.As shown in FIGS. 55-56 and 59, the combined sampling and separationdevice 6100 includes an upper housing 6102, a lower housing 6104, and aflexible pinch valve 6108 connecting the upper housing 6102 and lowerhousing 6104. As shown in FIGS. 57 and 58, the upper housing encloses anupper lytic chamber 6120, the lower housing encloses a lower separationchamber 6140, and the flexible pinch valve 6108 defines a fluid transferchannel 6130 therethrough. The upper lytic chamber 6120, fluid transferchannel 6130 and lower separation chamber 6140 can be orientated arounda central axis 6122.

The combined sampling and separation device 6100 further includes a pairof opposable compression tabs 6110, a valve actuator block 6106 andopposable actuator arms 6118 operable to “open” and “close” the flexiblepinch valve 6110. In operation, the valve actuator block 6106 can bemoved in a first direction (e.g., towards the compression tabs 6110, asrepresented by arrow 6107) to “open” the valve 6100. By moving theactuator block 6106 towards the compression tabs 6110 the actuator arms6118 push up the compression tabs 6110 moving the compression tabs 6110away from the flexible pinch valve thereby open the valve 6108. In theopen position, the fluid flow channel 6130 is opened allowing fluidcommunication between the upper lytic chamber 6120 and the lowerseparation chamber 6140 (as shown in FIG. 58). The valve actuator block6106 can also be moved in a second direction (e.g., away from thecompression tabs 6110, as represented by arrow 6109) to “close” thevalve 6108. When the actuator block 6106 is moved away from thecompression tabs 6110 the actuator arms 6118 move the pair of opposablecompression tabs 6110 to a “closed” position, thereby pinching closedthe flexible pinch valve 6108 (as shown in FIG. 60).

As shown in FIGS. 56-57 and 59, the combined sampling and separationdevice 6100 also includes a syringe needle 6112 for obtaining a samplefrom a specimen container, and a vacuum port 6114 for pulling a vacuumwithin the lytic chamber 6120, thereby assisting with loading of thedevice 6100. Optionally the syringe may further comprise a sheath (notshown) to protect the syringe needle from damage and/or contamination.Also, as shown in FIGS. 56-57 and 59, the combined sampling andseparation device 6100 includes a vacuum port 6114. The vacuum port willinclude a gas permeable filter or hydrophobic membrane 6116 that allowsgases to pass but prevents contamination. In operation, the vacuum portcan be connected to a pump (not shown) that can apply a vacuum to thesampling and separation device 6100 for the uptake of a sample from apositive specimen container.

As shown in FIGS. 58 and 60, the separation chamber 6140 includes anupper reservoir 6142, a middle tapered section 6144 and a lowercapillary tube 6146 all arranged around axis 6122 below the lyticchamber 6120. As shown, the middle tapered section 6144 connects thewider diameter upper reservoir 6142 and the smaller diameter capillarytube 6146. In one embodiment, the bottom wall 6150 of the capillary tube6146 is made of an optically transparent material for facilitatingoptical interrogation of a concentrated microbial agent (not shown)located at the bottom of the capillary tube 6146. In another embodiment,the separation device 6100 is made of an optically transparent materialto facilitate optical interrogation of a concentrated microbial agent(not shown) located at the bottom of the capillary tube 6146. As shown,the bottom wall 6150 opposite the capillary tube 6146 may be of areduced thickness to facilitate optical interrogation as indicated inFIGS. 58 and 60.

In operation, with the flexible pinch valve 6108 in the closed position,the lytic chamber 6120 can be loaded with a lysis buffer and a sampletaken from a positive culture container. In one embodiment, the lysisbuffer can be added to the lytic chamber 6120 of the separation device6100 using the syringe needle 6112. For example, the syringe needle 6112can be used to obtain an aliquot of lysis buffer (e.g., from a lysisbuffer reservoir), depositing the lysis buffer into the lytic chamber6120. Next, the syringe needle 6112 can be used to obtain a sample froma positive specimen container 500, depositing that sample into the lyticchamber 6120. The lysis buffer and sample are then mixed within thelytic chamber 6120, e.g., by agitation and/or vortexing of the samplingdevice 6100. The selective lysis step is allowed to proceed for asufficient time to allow the lysis reaction to be substantiallycompleted (e.g., from 1 to 5 minutes). This selective lysis stepselectively lyses undesired cells (i.e., non-microorganism cells) thatmay be present in the sample, e.g., blood cells and/or tissue cells. Inanother embodiment, the lytic chamber 6120 can be pre-loaded with alysis buffer and the sample loaded to the lytic chamber prior toagitation and/or vortexing. In still another embodiment, the samplingdevice 6100 can optionally be incubated to allow the selective lysisstep to proceed more quickly.

After the lysis step, the lysed sample and lysis buffer can betransferred to the separation chamber 6140 through the a fluid flowchannel 6130 for the separation of any microorganisms over a pre-loadeda density cushion, as described herein. To transfer the lysed sample andlysis buffer to the separation chamber 6140, the pair of opposablecompression tabs 6110 are moved to the open position, thereby openingthe flexible pinch valve 6108 and allowing fluid communication betweenthe lytic chamber 6120 and the separation chamber 6140 through the fluidflow channel 6130. With the flexible valve 6108 in the open position,the lysed sample and lysis buffer will flow via gravity through thefluid flow channel 6130 and onto the density cushion (not shown)contained in the separation chamber 6140. In one embodiment, by holdingthe separation device 6100 at an angle, the fluid can flow gently downthe interior wall of the separation chamber 6140 and onto the densitygradient.

The identification/characterization instrument 104 further includes aseparation and/or concentration station, optionally in the form of acentrifuge, which operates on the separation device 6100 so as toseparate the microbial agent from other products in the portion of thebiological sample and concentrate the microbial agent within theseparation device 6100. In one example, the microbial agent isconcentrated in the form of a pellet or pellet-like mass in the bottomof the capillary tube 6160 of the separation device 6100.

The identification/characterization instrument further includes aidentification and/or characterization module or read station (see,e.g., FIG. 1, 1918) which interrogates the concentrated microbial agentto identify and/or characterize the microbial agent as describedpreviously.

Another embodiment of the combined sampling and separation device 6300is shown in FIGS. 61-63. Like the combined sampling and separationdevice shown in FIGS. 56-60, the combined sampling and separation device6300 includes an upper housing 6302 enclosing a lytic chamber 6320, alower housing 6104 enclosing a separation chamber 6340, and a flexiblepinch valve 6308 defining therethrough a fluid transfer channel 6130.

The combined sampling and separation device 6300 further comprises apair of opposable compression tabs 6310, a valve actuator block 6306 andopposable actuator arms 6318 operable to “open” and “close” the flexiblepinch valve 6308. In operation, the valve actuator block 6306 can bemoved in a first direction (e.g., towards the compression tabs 6310, asrepresented by arrow 6307) to “open” the valve 6308. By moving theactuator block 6306 towards the compression tabs 6310 the actuator arms6318 push up the compression tabs 6310 moving the compression tabs 6310away from the flexible pinch valve thereby open the valve 6308. In theopen position, the fluid flow channel 6330 is opened allowing fluidcommunication between the upper lytic chamber 6320 and the lowerseparation chamber 6140 (as shown in FIG. 62). The valve actuator block6306 can also be moved in a second direction (e.g., away from thecompression tabs 6306) to “close” the valve 6308. When the actuatorblock 6306 is moved away from the compression tabs 6310 the actuatorarms 6318 move the pair of opposable compression tabs 6310 to a “closed”position, thereby pinching closed the flexible pinch valve 6308 (notshown).

As shown in FIGS. 61-63, the combined sampling and separation device6300 also comprises a syringe needle 6312 for obtaining a sample from apositive specimen container, a valve port 6314 for pulling a vacuumwithin the lytic chamber 6320, thereby assisting with loading of thedevice 6300. Optionally the syringe may further comprise a sheath (notshown) to protect the syringe needle from damage and/or contamination.The vacuum port will include a gas permeable filter or hydrophobicmembrane 6116 that allows gases to pass but prevents contamination. Thecombined sampling and separation device further comprises a vacuumchamber, which is optionally pre-charged with a vacuum and operableconnected to the sampling and separation device 6300 via a valve 6360 toapply a vacuum to the sampling and separation device 6300 for the uptakeof a sample from a positive specimen container.

Referring to FIG. 63, the valve mechanism 6360 of this embodimentcomprises a pump port 6370 that allows a pump (not shown) to operate theplunger 6380 between a vacuum position and a venting position. The valve6360 further comprises an interior chamber 6374, a vacuum port 6376, anda venting port 6378. In operation, the pump (not shown) can move theplunger to a first position or a vacuum position (as shown in FIG. 65)thereby opening a fluid communication channel from said valve port 6372,through the interior chamber 6374 and through the vacuum port 6376 tothe vacuum chamber 6362. In the first position or vacuum position, thevalve 6360 allows a vacuum to be applied to the sampling and separationdevice 6300, thereby controlling the uptake of a sample from positivespecimen container. The plunger 6380 can also be moved to a secondposition or venting position thereby opening a fluid communicationchannel from said valve port 6372, through the interior chamber 6374 andthrough the vacuum port 6378, thereby allowing the sampling andseparation device to vent a specimen container prior to the uptake of asample via the vacuum.

As shown in FIG. 62, the separation chamber 6340 may further comprise anupper reservoir 6342, a middle tapered section 6344 and a lowercapillary tube 6346 all arranged around axis 6322 below the lyticchamber 6320. As shown, the middle tapered section 6344 connects thewider diameter upper reservoir 6342 and the smaller diameter capillarytube 6346. In one embodiment, the bottom wall 6350 of the capillary tube6346 is made of an optically transport material for facilitating opticalinterrogation of a concentrated microbial agent (not shown) located atthe bottom of the capillary tube 6346. In another embodiment, theseparation device 6300 is made of an optically transparent material tofacilitate optical interrogation of a concentrated microbial agent (notshown) located at the bottom of the capillary tube 6346. As shown, thebottom wall 6350 opposite the capillary tube 6346 may be of a reducedthickness to facilitate optical interrogation as indicated in FIG. 62.

As one of skill in the art would appreciate, the sampling and separationdevice 6300 of this embodiment operates in a similar manner as thesampling and separation device 6100 of the first embodiment.Accordingly, a detailed description of the operation of this specificembodiment is excluded. After the lysis step has been carried out, thesampling and separation device 6300 of this embodiment can becentrifuged for separation and/or pelleting of any microorganismscontained therein. The sampling and separation device 6300 of thisembodiment may be pre-loaded with a lysis buffer and/or a densitycushion.

Referring now to FIGS. 64-67, a third embodiment of a combined samplingand separation device 6200 is shown. The combined sampling andseparation device 6200 includes an upper housing 6202, a lower housing6204, and a rotary connection 6206 connecting the upper housing 6202 andlower housing 6204. As shown in FIG. 65, the upper housing encloses anupper lytic chamber 6220, the lower housing encloses a lower separationchamber 6240, and the rotary connection 6206 defines a fluid transferchannel 6130 therethrough. The upper lytic chamber 6220, fluid transferchannel 6230 and lower separation chamber 6240 can be orientated arounda central axis 6222, as shown in FIG. 65.

In operation, the rotary connection 6206 can be rotated to an “open”position. In the open position, the fluid flow channel 6230 is openedallowing fluid communication between the upper lytic chamber 6220 andthe lower separation chamber 6240 (as shown in FIG. 65). The rotaryconnection 6206 can also be rotated to a “closed” position to close thefluid flow channel 6230. As shown in FIG. 65, the fluid flow channelcomprises an upper opening or channel 6232 through the upper portion ofthe rotary connection 6208 and a lower opening or channel 6234 throughthe lower portion of the rotary connection 6210. The rotary connection6206 of this embodiment may further comprise a sealing gasket 6218between the upper portion of the rotary connection 6208 and the lowerportion of the rotary connection 6210, as shown in FIG. 66, to preventleaks.

As shown in FIGS. 64-66, the combined sampling and separation device6200 also comprises a syringe needle 6212 for obtaining a sample from apositive specimen container, and a vacuum port 6214 for pulling a vacuumwithin the lytic chamber 6220, thereby allowing a sample to be loadinginto the lytic chamber 6260 of the device 6200. Optionally the syringemay further comprise a sheath (not shown) to protect the syringe needlefrom damage and/or contamination. The vacuum port 6214 will include agas permeable filter or hydrophobic membrane 6216 that allows gases topass but prevents contamination. In operation, the vacuum port 6214 canbe connected to a pump (not shown) that can apply a vacuum to thesampling and separation device 6200 for the uptake of a sample from apositive specimen container.

As shown in FIG. 65, the separation chamber 6240 may further comprise anupper reservoir 6242, a middle tapered section 6244 and a lowercapillary tube 6246 all arranged around axis 6222 below the lyticchamber 6220. As shown, the middle tapered section 6244 connects thewider diameter upper reservoir 6242 and the smaller diameter capillarytube 6246. In one embodiment, the bottom wall 6250 of the capillary tube6246 is made of an optically transparent material for facilitatingoptical interrogation of a concentrated microbial agent (not shown)located at the bottom of the capillary tube 6246. In another embodiment,the separation device 6200 is made of an optically transparent materialto facilitate optical interrogation of a concentrated microbial agent(not shown) located at the bottom of the capillary tube 6246. As shown,the bottom wall 6250 opposite the capillary tube 6246 may be of areduced thickness to facilitate optical interrogation as indicated inFIG. 65.

As one of skill in the art would appreciate, the sampling and separationdevice 6200 of this embodiment operates in a similar manner as thesampling and separation device 6100 of the first embodiment.Accordingly, a detailed description of the operation of this specificembodiment is excluded. After the lysis step has been carried out, thesampling and separation device 6200 of this embodiment can becentrifuged for separation and/or pelleting of any microorganismscontained therein. The sampling and separation device 6200 of thisembodiment may be pre-loaded with a lysis buffer and/or a densitycushion.

Referring now to FIGS. 67-69B, another embodiment of a combined samplingand separation device 6400 is shown. The combined sampling andseparation device 6400 includes an upper housing 6402, a lower housing6404, and a rotary valve 6406 connecting the upper housing 6402 andlower housing 6404. As shown in FIGS. 68B and 69B, the upper housingencloses an upper lytic chamber 6420, the lower housing encloses a lowerseparation chamber 6440, and the rotary valve 6306 defines a fluidtransfer channel 6430 therethrough. The upper lytic chamber 6420, fluidtransfer channel 6430 and lower separation chamber 6440 can beorientated around a central axis 6422, as shown in FIGS. 68B and 69B.

In operation, the rotary valve 6406 can be rotated via a valve handle6408 to an “open” position 6434 (see FIG. 79B). In the open position,the fluid flow channel 6430 is opened allowing fluid communicationbetween the upper lytic chamber 6420 and the lower separation chamber6440 (as shown in FIG. 69B). The rotary valve 6406 can also be rotatedto a “closed” position 6434 (see FIG. 68B) to close the fluid flowchannel 6430.

As shown in FIGS. 67-69B, the combined sampling and separation device6400 also comprises a syringe needle 6412 for obtaining a sample from apositive specimen container, and a vacuum port 6414 for pulling a vacuumwithin the lytic chamber 6420, thereby allowing a sample to be loadinginto the lytic chamber 6460 of the device 6400. Optionally the syringemay further comprise a sheath (not shown) to protect the syringe needlefrom damage and/or contamination. The vacuum port 6414 will include agas permeable filter or hydrophobic membrane 6416 that allows gases topass but prevents contamination. In operation, the vacuum port 6414 canbe connected to a pump (not shown) that can apply a vacuum to thesampling and separation device 6400 for the uptake of a sample from apositive specimen container.

As shown in FIGS. 68B and 69B, the separation chamber 6440 may furthercomprise an upper reservoir 6442, a middle tapered section 6444 and alower capillary tube 6446 all arranged around axis 6422 below the lyticchamber 6420. As shown, the middle tapered section 6444 connects thewider diameter upper reservoir 6442 and the smaller diameter capillarytube 6446. In one embodiment, the bottom wall 6450 of the capillary tube6446 is made of an optically transparent material for facilitatingoptical interrogation of a concentrated microbial agent (not shown)located at the bottom of the capillary tube 6446. In another embodiment,the separation device 6400 is made of an optically transparent materialto facilitate optical interrogation of a concentrated microbial agent(not shown) located at the bottom of the capillary tube 6446. As shown,the bottom wall 6450 opposite the capillary tube 6446 may be of areduced thickness to facilitate optical interrogation as indicated inFIGS. 68B and 69B.

As one of skill in the art would appreciate, the sampling and separationdevice 6400 of this embodiment operates in a similar manner as thesampling and separation device 6100 of the first embodiment.Accordingly, a detailed description of the operation of this specificembodiment is excluded. After the lysis step has been carried out, thesampling and separation device 6400 of this embodiment can becentrifuged for separation and/or pelleting of any microorganismscontained therein. The sampling and separation device 6400 of thisembodiment may be pre-loaded with a lysis buffer and/or a densitycushion.

From the above discussion, it will be appreciated that we have disclosedan apparatus for automated venting of a specimen container (500) havinga closure (e.g., septum) sealing the interior of the specimen containerfrom the environment, comprising:

a rack (e.g., FIG. 5, 1906) holding the specimen container 500;

a venting device (1902, FIG. 14, FIG. 32) having a needle 3202, achamber 3202 in fluid communication with the needle and a port 3208 influid communication with the chamber (FIGS. 14, 32, 43A); (Note: theventing device may take the form of one of the combined separation andsampling devices shown in FIGS. 51-59 particularly if configured with aneedle)

a robotic transfer mechanism (1910, FIGS. 1, 5, 247) moveable relativeto the rack;

a sample removal apparatus (1912, FIGS. 1, 5, 15, 17, 27, 37, 38),attached to the robotic transfer mechanism having gripping features forgripping the venting device;

wherein the sample removal apparatus and robotic transfer mechanism aremoveable relative to the specimen container so as to automaticallyinsert the needle of the venting device through the closure of thespecimen container to thereby vent the interior of the specimencontainer and obtain equilibrium between the interior of the specimencontainer and the atmosphere. (FIGS. 16, 38)

As discussed above, the rack is moveable between a first positionwherein the specimen container is oriented in an upward orientation(FIGS. 16, 17) and a second position wherein the specimen container isoriented in a downward orientation (FIGS. 18, 19), and wherein theinsertion of the needle of the venting device occurs while the rack ismoved to the first position (FIGS. 16, 17).

As shown in the embodiment of FIG. 32A, the venting device 1902 mayinclude a hydrophobic filter 3218 positioned within the venting deviceproximate to the port.

The apparatus further includes a pneumatic system 1914 (FIG. 1) coupledto the port 3208 of the venting device, e.g., as shown in FIGS. 14 and34 (via tube 3402 connected to the vacuum pump 1710 of FIG. 31). Thus,the pneumatic system 1910 may include a tube 3402 having a first endconnected to the port of the venting device (see FIG. 34) and a secondend connected to a pneumatic pump (FIG. 36).

As discussed above, in some embodiments the venting device 1902 isloaded with a selective lytic agent (see FIG. 14). In some embodimentsthe venting device 1902 includes a sheath covering the needle (see FIG.32, 43, sheath 3214). The sheath can be flexible (see FIGS. 44-46) andthe flexible sheath covers a portion of the needle external to thespecimen container during the venting of the specimen container (FIG.38).

As noted above, the robotic transfer mechanism 1910 can take a varietyof forms, including a multi-axis robot arm (see FIGS. 31, 37).Alternatively, the robotic transfer mechanism can take the form of aCartesian robot moveable in at least two orthogonal directions relativeto the rack 1906 (see FIG. 5). As shown in FIGS. 5 and 35, the sampleremoval apparatus can include gripping fingers (See 1956, FIGS. 34, 35).As shown in FIG. 34, the sample removal apparatus includes a slide 1952coupled to the first end of the tube 3402, the slide moveable relativeto the venting device 1902 to advance the first end of the tube 3402over the port 3208 of the venting device 1902.

It will also be appreciated that we have described an apparatus forautomated sampling of a specimen container having a closure sealing theinterior of the specimen container from the environment, comprising: arack 1906 holding the specimen container; a sampling device 1902 havinga needle, a chamber in fluid communication with the needle and a port influid communication with the chamber; a robotic transfer mechanism 1910moveable relative to the rack; a sample removal apparatus 1912 attachedto the robotic transfer mechanism having gripping features for grippingthe sampling device 1902; and a pneumatic system 1914 coupled to theport of the sampling device (e.g., via tube 3402); wherein the sampleremoval apparatus and robotic transfer mechanism are moveable so as toautomatically insert the needle of the sampling device through theclosure of the specimen container, the pneumatic system operative todraw a portion of a sample contained within the specimen container intothe chamber of the sampling device via the needle. (FIGS. 15, 18, 19,37, 38). (Note: the sampling device may take the form of one of thecombined separation and sampling devices shown in FIGS. 51-69).

In one embodiment, the sample removal apparatus 1912 first inserts theneedle 3202 into the specimen container while the specimen container isrotated to first, upward position to thereby vent the specimen container500 and then withdraws the sample from the specimen container after therack is rotated to a second, downward position.

An automated method for venting a specimen container in the form of abottle having a closure sealing the interior of the specimen containerfrom the environment has been disclosed comprising the steps of: holdingthe bottle 500 in a rack 1906 (FIG. 5); automatically and with the aidof robotic apparatus grasping a venting device having a needle, achamber connected to the needle and a port connected to the chamber(FIGS. 14, 15); automatically moving the robotic apparatus so as toplace the venting device in a position proximate to the bottle in therack (FIGS. 16, 17); automatically inserting the needle of the ventingdevice through the closure so as to place the needle into the interiorof the bottle (FIG. 17), and bringing the interior of the specimencontainer to equilibrium with the atmosphere via the needle, chamber andport.

The method may include a step of moving the rack so as to place thebottle into an upward orientation and performing the automatic insertingstep while the bottle is in the upward orientation, as shown in FIGS. 16and 17. The method may also include the steps of moving the rack so asto place the bottle into a downward orientation; applying vacuum to theport of the sampling device; and drawing a portion of a sample containedwithin the bottle into the sampling device. (FIGS. 18 and 19).

In one possible configuration, the sampling device 1902 is loaded with aselective lytic agent prior to the drawing step (see FIG. 14, the device1902 is either pre-loaded with the lytic agent or else the lytic agentis added from a container 1802 within the instrument 104, see FIG. 27,e.g., using the vacuum function of the pneumatic system 1914 and tubing3402).

In another aspect, an automated method for sampling a specimen containerin the form of a bottle 500 having a closure sealing the interior of thespecimen container from the environment is disclosed, comprising thesteps of: (a) holding the bottle in a rack 1906; (b) automatically andwith the aid of robotic apparatus 1910/1912 grasping a sampling device1902 having a needle 3202 (FIG. 14, 32), a chamber 3204 connected to theneedle and a port 3208 connected to the chamber; (c) automaticallymoving the robotic apparatus so as to place the sampling device in aposition proximate to the bottle in the rack (FIGS. 6, 17); (d)automatically inserting the needle of the sampling device through theclosure so as to place the needle into the interior of the bottle (FIG.18-19, 38), and (e) applying vacuum to the port of the sampling deviceso as to draw a portion of a sample contained in the bottle into thesampling device.

The method may include a step of moving the rack so as to place thebottle into an downward orientation and performing the automaticinserting step while the bottle is in the downward orientation. (FIG.18, 19). The method may further include the steps of moving the rack soas to place the bottle into an upward orientation; and wherein the stepof inserting the needle of the sampling device is performed while thebottle is in the upwards orientation; and wherein the method furthercomprises the step of venting the bottle to atmosphere prior to the stepof applying vacuum to the port of the sampling device. (FIGS. 16, 17).

In one embodiment, the method includes a step of loading the samplingdevice with a selective lytic agent prior to the step of inserting theneedle of the sampling device through the closure of the specimencontainer, e.g., from a container 1802 of selective lytic agent (FIG.27) at the time of use.

The sampling method may be repeated more than one time. Thus, in oneaspect, the method may include the steps of repeating steps (b), (c),(d) and (e) more than one time on the same bottle.

As shown in FIGS. 44-46 the method may further comprise the step ofinjecting some or all of the portion of the sample within the samplingdevice into a disposable separation device 1902 used for separation andconcentration of a microbial agent present in the sample.

The sampling method further include the step of lysing cellularcomponents of the sample within the separation device 1904. The methodmay also include the step of centrifuging the sampling device 1902 toseparate and concentrate a microbial agent within the sampling device.(As note above, the sampling device may take the form of one of thecombined separation and sampling devices shown in FIGS. 51-69).

The sampling method may further include the step of incubating thebottle while the bottle is held in the rack, e.g., in the embodimentshown in FIG. 27-29 where the rack is positioned within an incubationenclosure 1812.

As used herein, the term “needle” is intended to be interpreted broadlyto refer to a hollow spike-like element. That is, the needle 3202 (FIGS.14, 33) on the sampling and/or venting device 1902 could take a varietyof forms such as a plastic hollow spike.

Presently preferred and alternative embodiments of the inventiveautomated identification and/or characterization instrument have beendescribed with particularity. However, persons skilled in the art willunderstand that variation from the details of the disclosed embodimentsmay be made. All questions concerning the scope of the invention are tobe answered by reference to the appended claims.

1. Apparatus for automated sampling of a specimen container having aclosure sealing the interior of the specimen container from theenvironment, comprising: a rack holding the specimen container; asampling device having a needle, a chamber in fluid communication withthe needle and a port in fluid communication with the chamber; a robotictransfer mechanism moveable relative to the rack; a sample removalapparatus attached to the robotic transfer mechanism having grippingfeatures for gripping the sampling device; and a pneumatic systemcoupled to the port of the sampling device; wherein the sample removalapparatus and robotic transfer mechanism are moveable so as toautomatically insert the needle of the sampling device through theclosure of the specimen container, the pneumatic system operative todraw a portion of a sample contained within the specimen container intothe chamber of the sampling device via the needle.
 2. The apparatus ofclaim 1, wherein the rack is moveable between a first position whereinthe specimen container is oriented in an upward orientation and a secondposition wherein the specimen container is oriented in a downwardorientation, and wherein the insertion of the needle of the samplingdevice occurs while the rack is moved to the first position.
 3. Theapparatus of claim 1, wherein the disposable sampling device furthercomprises a hydrophobic filter positioned within the sampling deviceproximate to the port.
 4. The apparatus of claim 1, wherein thepneumatic system comprises a tube having a first end connected to theport of the sampling device and a second end connected to a pneumaticpump.
 5. The apparatus of claim 1, wherein the sampling device is loadedwith a selective lytic agent.
 6. The apparatus of claim 1, wherein thesampling device further comprises a sheath covering the needle.
 7. Theapparatus of claim 6, wherein the sheath comprises a flexible sheath andwherein the flexible sheath covers a portion of the needle exterior tothe specimen container during the drawing of the sample into thesampling device.
 8. The apparatus of claim 1, wherein the robotictransfer mechanism comprises a multi-axis robot arm.
 9. The apparatus ofclaim 1, wherein the robotic transfer mechanism comprises a Cartesianrobot moveable in at least two orthogonal directions relative to therack.
 10. The apparatus of claim 2, wherein the sample removal apparatusfirst inserts the needle into the specimen container while the specimencontainer is rotated to the first position to thereby vent the specimencontainer and then withdraws the sample from the specimen containerafter the rack is rotated to the second position.
 11. An automatedmethod for venting a specimen container in the form of a bottle having aclosure sealing the interior of the specimen container from theenvironment, comprising the steps of: holding the bottle in a rack;automatically and with the aid of robotic apparatus grasping a ventingdevice having a needle, a chamber connected to the needle and a portconnected to the chamber; automatically moving the robotic apparatus soas to place the venting device in a position proximate to the bottle inthe rack; automatically inserting the needle of the venting devicethrough the closure so as to place the needle into the interior of thebottle, and bringing the interior of the specimen container toequilibrium with the atmosphere via the needle, chamber and port. 12.The method of claim 11, further comprising the step of moving the rackso as to place the bottle into an upward orientation and performing theautomatic inserting step while the bottle is in the upward orientation.13. The method of claim 12, further comprising the steps of: moving therack so as to place the bottle into a downward orientation; applyingvacuum to the port of the sampling device; and drawing a portion of asample contained within the bottle into the sampling device.
 14. Anautomated method for sampling a specimen container in the form of abottle having a closure sealing the interior of the specimen containerfrom the environment, comprising the steps of: (a) holding the bottle ina rack; (b) automatically and with the aid of robotic apparatus graspinga sampling device having a needle, a chamber connected to the needle anda port connected to the chamber; (c) automatically moving the roboticapparatus so as to place the sampling device in a position proximate tothe bottle in the rack; (d) automatically inserting the needle of thesampling device through the closure so as to place the needle into theinterior of the bottle, and (e) applying vacuum to the port of thesampling device so as to draw a portion of a sample contained in thebottle into the sampling device.
 15. The method of claim 14, furthercomprising the step of moving the rack so as to place the bottle into andownward orientation and performing the automatic inserting step whilethe bottle is in the downward orientation.
 16. The method of claim 14,further comprising the steps of: moving the rack so as to place thebottle into an upward orientation; and wherein the step of inserting theneedle of the sampling device is performed while the bottle is in theupwards orientation; and wherein the method further comprises the stepof venting the bottle to atmosphere prior to the step of applying vacuumto the port of the sampling device.
 17. The method of claim 14, furthercomprising the step of loading the sampling device with a selectivelytic agent prior to the step of inserting the needle of the samplingdevice through the closure of the specimen container.
 18. The method ofclaim 14, further comprising the steps of repeating steps (b) (c) (d)and (e) more than one time on the same bottle.
 19. The method of claim14, further comprising the step of injecting some or all of the portionof the sample within the sampling device into a disposable separationdevice.
 20. The method of claim 14, further comprising the step oflysing cellular components of the sample within the disposable samplingdevice.
 21. The method of claim 20, further comprising the step ofcentrifuging the sampling device to separate and concentrate a microbialagent within the sampling device.